Riboflavin synthase genes and enzymes and methods of use

ABSTRACT

Through function complementation of  E. coli  auxotrophs, the ultimate and pentultimate enzymes of the spinach riboflavin biosynthetic pathway have been cloned, namely, lumazine synthase (LS) and riboflavin synthase (RS). This invention relates to the isolation of nucleic acid fragments from plants or fungi that encode LS protein. The invention also relates to the isolation of nucleic acid fragments from plants or fungi that encode RS protein. In addition, the invention also relates to the construction of chimeric genes encoding all of a portion of LS, in sense or antisense orientation, wherein the expression of the chimeric gene results in production of altered levels of plant LS in a transformed host cell. Furthermore, the invention also relates to the construction of chimeric genes encoding all of a portion of RS, in sense or antisense orientation, wherein the expression of the chimeric gene results in production of altered levels of plant or fungal RS in a transformed host cell. In vivo and in vitro methods to identify herbicide or fungicide candidates are included that evaluate the ability of a chemical compound to inhibit the activity of a plant or fungal LS enzyme or a plant or fungal RS enzyme.

This is a Continuation-in-part application, U.S. Ser. No. 08/181,183,filed Jan 13, 1994, now abandoned which is a Continuation-In-Part U.S.Ser. No. 08/912,218, filed Aug. 15, 1997 now abandoned.

FIELD OF THE INVENTION

This invention is in the field of plant and fungal molecular biology.More specifically, this invention pertains to nucleic acid fragmentsencoding proteins involved in the riboflavin biosynthetic pathway ofplants or fungi.

BACKGROUND OF THE INVENTION

Riboflavin, vitamin B₂, is the precursor of flavin mononucleotide (FMN)and flavin adenine dinucleotide (FAD), essential cofactors for a numberof mainstream metabolic enzymes that mediate hydride, oxygen, andelectron transfer reactions. Riboflavin-dependent enzymes includesuccinate dehydrogenase, NADH dehydrogenase, ferredoxin-NADP⁺oxidoreductase, acyl-CoA dehydrogenase, and the pyruvate dehydrogenasecomplex. Consequently, fatty acid oxidation, the TCA cycle,mitochondrial electron-transport, photosynthesis, and numerous othercellular processes are critically dependent on either FMN or FAD asprosthetic groups. Other notable flavoproteins include glutathionereductase, glycolate oxidase, P450 oxido-reductase, squalene epoxidase,dihydroorotate dehydrogenase, and α-glycerophosphate dehydrogenase.Genetic disruption of riboflavin biosynthesis in E. coli (Richter etal., J. Bacteriol. 174:4050-4056 (1992)) and S. cerevisiae (Santos etal., J. Biol. Chem. 270:437-444 (1995)) results in a lethal phenotypethat is only overcome by riboflavin supplementation. This is notsurprising, considering the ensemble of deleterious pleiotropic effectsthat would occur with riboflavin deprivation.

Riboflavin is synthesized by plants and numerous microorganisms,including bacteria and fungi (Bacher, A., Chemistry and Biochemistry ofFlavoproteins (Müller, F., ed.) vol. 1, pp. 215-259, Chemical RubberCo., Boca Raton, Fla. (1990)). Since birds, mammals, and other higherorganisms are unable to synthesize the vitamin and, instead, rely on itsdietary ingestion to meet their metabolic needs, the enzymes that areresponsible for riboflavin biosynthesis are potential targets for futureantibiotics, fungicides, and herbicides. Moreover, it is possible thatthe distantly-related plant and microbial enzymes have distinctcharacteristics that could be exploited in the development of potentorganism-specific inhibitors. Thus, a detailed understanding of thestructure, mechanism, kinetics, and substrate-binding properties of theriboflavin biosynthetic enzyme(s), from plants for example, would serveas a starting point for the rational design of chemical compounds thatmight be useful as herbicides. Having the authentic plant protein(s) inhand would also provide a valuable tool for the in vitro screening ofchemical libraries in search of riboflavin biosynthesis inhibitors.

Bacterial and fungal riboflavin biosynthesis has been intensivelystudied for more than four decades (For recent reviews, see Bacher, A.,Chemistry and Biochemistry of Flavoproteins (Müller, F., ed.) vol. I,pp. 215-259 and 293-316 Chemical Rubber Co., Boca Raton, Fla. (1990)).The synthetic pathway consists of seven distinct enzyme catalyzedreactions, with guanosine 5′-triphosphate (GTP) and ribulose 5-phosphatethe ultimate precursors. While the second and third steps of riboflavinbiosynthesis occur in opposite order in bacteria and fungi, theremaining pathway intermediates are identical in both microorganisms.Structurally and mechanistically, the last two reactions in the pathway,namely, those catalyzed by 6,7-dimethyl-8-ribityllumazine synthase (LS)and riboflavin synthase (RS), are best characterized. In B. subtilis,these two enzymes are physically associated with each other in a hugespherical particle with a combined molecular mass of about 1 MDa (Bacheret al., J. Biol Chem. 255:632-637 (1980); Ritsert et al., J. Mol. Biol.253, 151-167 (1995); Bacher et al., Biochem. Soc. Trans. 24(1):89-94(1996)); the X-ray structure of the bifunctional protein complex hasbeen determined at 3.3 angstrom resolution (Ladenstein et al., J. Mol.Biol 203:1045-1070). The LS/RS complex consists of 60 LS subunits thatare organized into 12 pentamers to form a hollow icosahedral capsid.Encaged in the central core of this structure resides a single moleculeof RS, a trimer of three identical subunits. Kinetic studies reveal thatthe compartmentation of the two enzymes within the complex improves theoverall catalytic efficiency of riboflavin production at low substrateconcentrations, presumably via “substrate channeling” (Kis et al., J.Biol. Chem. 270:16788-16795 (1995)). Although a bifunctional LS/RScomplex has not been observed in other microorganisms, it was recentlyshown that the native E. coli LS also exists in vivo as a hollowicosahedral capsid of 60 identical subunits (Mörtl et al., J. Biol.Chem. 271:33201-33207 (1996)).

LS, the penultimate enzyme of riboflavin biosynthesis, catalyzes thecondensation of 3,4-dihydroxy-2-butanone 4-phosphate with4-ribitylamino-5-amino-2,6-dihydroxypyrimidine (RAADP) to yield 1 moleach of orthophosphate and 6,7-dimethyl-8-(1′-D-ribityl)-lumazine(DMRL). The latter is the immediate precursor of riboflavin. LS-encodinggenes have been cloned from numerous microorganisms, including E. coli(Taura et al., Mol. Gen. Genet. 234:429-432 (1992)), A. pleuropneumoniae(Fuller et al., J. Bacteriol. 177:7265-7270 (1995)), P. phosphoreum (Leeet al., J. Bacteriol. 176:2100-2104 (1994)), B. subtilis (Mironov etal., Dokl. Akad Nauk SSSR 305:482-487 (1989)), and S. cerevisiae(Garcia-Ramirez et al., J. Biol. Chem. 270:23801-23807 (1995)). In allcases, the subunit molecular mass of the LS gene product is small,ranging in size from ˜16-17 kDa.

While the various LS homologs all share certain structural features incommon, their overall homology at the primary amino acid sequence levelis rather poor. For example, as determined with the Genetics ComputerGroup Gap program (Wisconsin Package Version 9.0, Genetics ComputerGroup (GCG), Madison, Wis.), the E. coli LS is only 58%, 65%, 53%, and36% identical to the homologous proteins of A. pleuropneumoniae, P.phosphoreum, B. subtilis and S. cerevisiae, respectively. Indeed,pairwise comparisons of these five proteins reveal that the two mostsimilar homologs share only 72% identity.

The terminal step of riboflavin biosynthesis is mediated by RS. Thisenzyme catalyzes the dismutation of two molecules of DMRL to yield 1 molof riboflavin and RAADP. That the latter product is also one of thesubstrates of LS explains in part the enhanced catalytic efficiency ofthe B. subtilis LS/RS complex noted above. Although the crystalstructure of RS remains to be determined, it is surmised that the nativebacterial (Bacher et al., J. Biol. Chem. 255:632-637 (1980)) and fungal(Santos et al., J. Biol. Chem. 270:437-444 (1995)) proteins are trimers,each consisting of three identical ˜25 kDa subunits. To date, RS hasonly been cloned from about a dozen microorganisms, and all of thespecies that have been examined exhibit marked internal homology intheir N-terminal and C-terminal domains (Schott et al., J. Biol. Chem.265:4204-4209 (1990); Santos et al., J. Biol. Chem. 270:437-444 (1995)).Based on these observations, it has been suggested that the two halvesof the RS protomer have arisen through gene duplication, and that eachcontains a substrate-binding site for DMRL.

Despite this structural similarity, however, the overall sequencehomology of the various RS proteins is extremely limited. Thus, the E.coli RS protein is only 32%, 36%, 35%, and 31% identical to itscounterparts in S. cerevisiae, P. phosphoreum., B. subtilis, and P.leiognathi; the GenBank accession numbers for the latter four proteinsare Z21621, L11391, X51510 and M90094, respectively.

With the exception of GTP cyclohydrolase II, the first committed enzymeof riboflavin biosynthesis, virtually nothing is known about theriboflavin biosynthetic machinery of higher plants. The gene for thisprotein was recently cloned from an arabidopsis cDNA library (Kobayashiet al., Gene 160:303-304 (1995)). The protein sequence of the clonedplant gene is only 37-58% identical to the homologous proteins from E.coli, B. subtilis, P. leiognathi, and P. phosporeum. While full-lengthcDNA sequences have not been reported for any other plant riboflavinbiosynthetic enzyme, the GenBank database contains two ESTs (ExpressedSequence Tags) that potentially correspond to plant LS genes. One ofthese is from castor bean and the other is from arabidopsis.

The castor bean cDNA clone (GenBank accession number T15152; van de Looet al., Plant Physiol. 108:1141-1150 (1995)) is truncated at its 5′ end,and is missing DNA corresponding to at least 60 N-terminal amino acidresidues. The arabidopsis cDNA clone (GenBank accession number Z34233;direct submission) was identified through a BLAST (Basic Local AlignmentSearch Tool; Altschul et al., J. Mol. Biol. 215:403-410 (1990)) searchusing the TBLASTN algorithm provided by the National Center forBiotechnology Information (NCBI). The query sequence for the BLASTsearch was the translated E. coli LS gene (GenBank accession numberX64395) and the probability score for similarity to the arabidopsis ESTwas P=0.45. Unfortunately, the portion of the cDNA insert that wassequenced contained only the last 26 C-terminal amino acid residues ofthe protein, so it is not known whether it is a partial or full-lengthcDNA clone. Since neither of these clones possess a polyA tail, it ispossible that they reflect contaminating microbial DNA that wasintroduced at some point during the preparation of the cDNA libraries.

In contrast to LS, BLAST searches failed to identify any plant DNAsequences in the GenBank database with significant primary amino acidsequence homology to either E. coli or yeast RS. However, RS activityhas been detected in extracts from various plant species (Plaut, G.,Metabolic Pathways (Greenberg, D. M., ed.), vol II, p. 673, AcademicPress, New York, (1961)), and partial purification of the spinachhomolog has been described (Mitsuda et al., Methods Enzymol. 18b:539-543(1970)).

From the foregoing discussion, it is apparent that too little is knownabout plant LS or RS genes/proteins and their relationship to knownmicrobial homologs to allow isolation of LS- or RS-encoding genes fromany plant species using most classical approaches. The latter includehybridization probing of cDNA libraries with homologous or heterologousgenes, PCR-amplification of the gene of interest using oligionucleotideprimers corresponding to conserved amino acid sequence motifs, and/orimmunological detection of expressed cDNA inserts in microbial hosts.Unfortunately, these techniques would not be expected to be very usefulfor the isolation of plant LS or RS genes, since they all heavily relyon the presence of significant structural similarity (i.e., DNA or aminoacid sequence) with known proteins and genes that have the samefunction. Given the observation that LS and RS proteins are both sopoorly conserved, even amongst microorganisms, it is highly unlikelythat the known microbial homologs would share significant structuralsimilarities with their counterparts in higher plants.

An alternative approach that has been used to clone biosynthetic genesin other metabolic pathways from higher eucaryotes is throughcomplementation of microbial mutants that are deficient in the enzymeactivity of interest. Since this strategy relies only on the functionalsimilarity between the protein encoded for by the disrupted host geneand the target gene of interest, it is ideally suited for cloningstructurally dissimilar proteins that catalyze the same reaction. Forfunctional complementation, a cDNA library is constructed in a vectorthat can direct the expression of the cDNA in the microbial host. Theplasmid library is then introduced into the mutant microbe, and coloniesare selected that are no longer phenotypically mutant. Indeed, the LS(García-Ramirez et al., J. Biol. Chem. 270:23801-23807 (1995)) and RS(Santos et al., J. Biol. Chem. 270:437-444 (1995) of yeast, andarabidopsis GTP cyclohydrolase II (Kobayashi et al, Gene 160:303-304(1995)) were all cloned through functional complementation of microbialriboflavin auxotrophs. This strategy has also worked for isolating genesfrom higher eucaryotes that are involved in other metabolic pathways,including lysine biosynthesis (Frisch et al., Mol. Gen. Genet.228:287-293 (1991)), purine biosynthesis (Aimi et al., J. Biol. Chem.265:9011-9014 (1990)), and tryptophan biosynthesis (Niyogi et al., PlantCell 5:1011-1027 (1993)), and has also been successfully employed in theisolation of various plant genes including glutamine synthetase (Snustadet al., Genetics 120:1111-1124 (1988)), pyrroline-5-carboxylatereductase (Delauney et al., Mol. Genet. 221:299-305 (1990)),dihydrodipicolinate synthase (Frisch et al., Mol Gen. Genet. 228:287-293(1991)), 3-isopropylmalate dehydrogenase (Ellerstrom et al., Plant Mol.Biol. 18:557-566 (1992)), and dihydroorotate dehydrogenase (Minet etal., Plant J. 2:417-422 (1992)).

Despite the obvious attractive features of cloning by functionalcomplementation, there are several reasons why this approach might notwork when applied to the higher plant LS and RS genes. First, theeucaryotic cDNA sequence might not be expressed at adequate levels inthe mutant microbe for a variety of reasons, including differences inpreferred codon usage. Second, the cloned eucaryotic gene might notproduce a functional polypeptide, if for instance, enzyme activityrequires a post-translational modification, such as acetylation,glycosylation, or phosphorylation that is not carried out by themicrobial host. Third, the heterologous plant protein might be lethal tothe host, thus rendering its expression impossible. Fourth, theeucaryotic protein might fail to achieve its native conformation in theforeign microbial environment, due to folding problems, inclusion bodyformation, or various other reasons. It is also possible that the higherplant LS and RS enzymes are nuclear-encoded proteins that areposttranslationally targeted to chloroplasts, mitochondrial, or someother organelle that is not present in the microbial host. If this werethe case and proteolytic removal of the organellar targeting sequencewas required for enzyme activity, cloning these genes by functionalcomplementation would not be possible.

SUMMARY OF THE INVENTION

The instant invention relates to isolated nucleic acid fragmentsencoding plant or fungal enzymes involved in riboflavin biosynthesis.Specifically, this invention concerns isolated nucleic acid fragmentsencoding a plant or fungal LS, wherein the plant is spinach, tobacco orarabidopsis and the fungus is Magnaporthe grisea. This invention alsoconcerns isolated nucleic acid fragments encoding a plant or fungal RS,wherein the plant is spinach or arabidopsis and the fungus isMagnaporthe grisea. In addition, this invention relates to nucleic acidfragments that are complementary to nucleic acid fragments encoding aplant or fungal LS enzyme or a plant or fungal RS enzyme.

Specific isolated nucleic acid fragments encoding a plant LS enzyme are(a) an isolated nucleic acid fragment encoding all or a substantialportion of the amino acid sequence selected from the group consisting ofSEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6; (b) an isolated nucleic acidfragment that is substantially similar to an isolated nucleic acidfragment encoding all or a substantial portion of the amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4and SEQ ID NO:6; (c) an isolated nucleic acid fragment encoding apolypeptide having at least 72% identity with the amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQID NO:6; and (d) an isolated nucleic acid fragment that is complementaryto (a), (b) or (c).

Special isolated nucleic acid fragments encoding a plant RS enzyme are(a) an isolated nucleic acid fragment encoding all or a substantialportion of the amino acid sequence selected from the group consisting ofSEQ ID NO:8 and SEQ ID NO:10; (b) an isolated nucleic acid fragment thatis substantially similar to an isolated nucleic acid fragment encodingall or a substantial portion of the amino acid sequence selected fromthe group consisting of SEQ ID NO:8 and SEQ ID NO:10; (c) an isolatednucleic acid fragment encoding a polypeptide having at least 70%identity with the amino acid sequence selected from the group consistingof SEQ ID NO:8 and SEQ ID NO:10; and (d) an isolated nucleic acidfragment that is complementary to (a), (b) or (c).

Specific isolated nucleic acid fragments encoding a fungal RS enzyme are(a) an isolated nucleic acid fragment encoding all or a substantialportion of the amino acid sequence set forth in SEQ ID NO:12; (b) anisolated nucleic acid fragment that is substantially similar to anisolated nucleic acid fragment encoding all or is homologous to at leasta substantial portion of the amino acid sequence set forth in SEQ IDNO:12; (c) an isolated nucleic acid fragment that is complementary to(a) or (b).

Specific isolated nude acid fragments encoding a fungal LS enzyme are(a) an isolated nucleic acid fragment encoding all or a substantialportion of the amino acid sequence set forth in SEQ ID NO:38; (b) anisolated nucleic acid fragment that is substantially similar to anisolated nucleic acid fragment encoding all or is homologous to at leasta substantial portion of the amino acid sequence set forth in SEQ IDNO:38; and (c) an isolated nucleic acid fragment that is complementaryto (a) or (b).

In another embodiment, the instant invention relates to chimeric genesencoding a plant or fungal LS enzyme or a plant or fungal RS enzyme orto chimeric genes that comprise nucleic acid fragments that arecomplementary to the nucleic acid fragments encoding the enzymes,operably linked to suitable regulatory sequences, wherein expression ofthe chimeric genes results in production of levels of the encodedenzymes in transformed host cells that are altered (i.e., increased ordecreased) from the levels produced in the untransformed host cells.

In a further embodiment, the instant invention concerns a transformedhost cell comprising in its genome a chimeric gene encoding a plant orfungal LS enzyme or a plant or fungal RS enzyme, operably linked tosuitable regulatory sequences, wherein expression of the chimeric generesults in production of altered levels of a plant LS enzyme or a plantor fungal RS enzyme in the transformed host cell. The transformed hostcells can be of eucaryotic or procaryotic origin, and include cellsderived from higher plants and microorganisms. The invention alsoincludes transformed plants that arise from transformed host cells ofhigher plants, and from seeds derived from such transformed plants.

An additional embodiment of the instant invention, concerns a method ofaltering the level of expression of a plant or fungal LS enzyme or aplant or fungal RS enzyme in a transformed host cell comprising: a)transforming a host cell with the chimeric gene encoding a plant orfungal LS enzyme or a plant or fungal RS enzyme, operably linked tosuitable regulatory sequences; and b) growing the transformed host cellunder conditions that are suitable for expression of the chimeric genewherein expression of the chimeric gene results in production of alteredlevels of LS or RS in the transformed cell.

An additional embodiment of the instant invention concerns a method forobtaining a nucleic acid fragment encoding all or substantially all ofan amino acid sequence encoding a plant or fungal LS enzyme or a plantor fungal RS enzyme.

Additionally, an in vivo method is provided for identifying as anherbicidal or fungicidal candidate a chemical compound that inhibits theactivity of a plant or fungal LS enzyme or a plant or fungal RS enzymeand thus serve as a crop protection chemical comprising the steps of:(a) disrupting the endogenous LS or RS gene of a suitable microbialhost, rendering growth of the microbial host cell dependent on addedriboflavin; (b) transforming the altered microbial host cell of step (a)with a chimeric gene comprising an isolated nucleic acid fragmentencoding a plant or fungal LS enzyme or a plant or fungal RS enzyme, thechimeric gene operably linked to at least one suitable regulatorysequence that allows its expression in the microbial host cell; (c)growing the transformed host cell of step (a) under conditions suitablefor expression of the chimeric gene plant or fungal LS or RS gene; (d)contacting the transformed microbial host cell with a chemical compoundof interest in a well-controlled experiment while the host cell isgrowing exponentially and in both the presence and absence of addedriboflavin; (e) identifying as an herbicide or fungicide candidate thechemical compound of interest that inhibits growth of the transformedmicrobial host cell only when grown in the absence of added riboflavin.Suitable isolated nucleic acid fragments are those set out above.Suitable microbial hosts for this in vivo assay include the E. coli LSand RS riboflavin auxotrophs that are described below, both of whichnormally require riboflavin supplementation for growth. Specificinhibition of the functional plant or fungal genes that are introducedinto these mutants could then be assessed directly in parallel assays inwhich the transformed host cells are grown in both the presence andabsence of added riboflavin. Those inhibitory compounds that only affectmetabolic activity (growth) in the absence of riboflavin supplementationrepresent potential herbicides and/or fungicides.

In an alternate embodiment, an in vitro method is provided foridentifying as an herbicide or fungicide candidate a chemical compoundthat inhibits the activity of a plant or fungal LS enzyme or a plant orfungal RS enzyme and thus serve as a crop protection chemical comprisingthe steps of: (a) transforming a host cell with a chimeric genecomprising a nucleic acid fragment encoding a plant or fungal LS enzymeor a plant or fungal RS enzyme, the chimeric gene operably linked to atleast one suitable regulatory sequence; (b) growing the transformed hostcell of step (a) under conditions suitable for expression of thechimeric gene resulting in the production of the plant or fungal LSenzyme or a plant or fungal RS enzyme; (c) purifying the plant or fungalLS enzyme or the plant or fungal RS enzyme expressed by the transformedhost cell; (d) contacting the enzyme with a chemical compound ofinterest; and (e) identifying as an herbicide or fungicide candidate thechemical compound of interest that reduces the activity of the plant orfungal LS enzyme or plant or fungal RS enzyme relative to the activityof the respective enzyme in the absence of the chemical compound ofinterest. Such reduced activity indicates that the chemical compound ispotentially useful as a crop protection chemical. Suitable isolatednucleic acid fragments are those set out above.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

FIGS. 1-4 show primary amino acid sequence alignments generated with theGCG Pileup program (Genetics Computer Group, Madison, Wis.) using thegap creation default value of 12, and the gap extension default value of4.

FIG. 1 shows the primary amino acid sequence alignments of knownmicrobial RS homologs and the cloned spinach RS precursor protein.

FIG. 2 shows the primary amino acid sequence alignments of knownmicrobial LS homologs and the cloned spinach LS precursor protein.

FIG. 3 shows the primary amino acid sequence alignments of spinach andarabidopsis RS precursor proteins. Boxed residues denote the putativechloroplast targeting sequences (e.g., transit peptides).

FIG. 4 shows primary amino acid sequence alignments of spinach, tobaccoand arabidopsis LS precursor proteins. Boxed residues denote theputative chloroplast targeting sequences (e.g., transit peptides).

The following sequence descriptions and sequence listings attachedhereto comply with the rules governing nucleotide and/or amino acidsequence disclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825. The Sequence Descriptions contain the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IYUB standards describedin Nucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219(2):345-373 (1984) which are herein incorporated byreference. The present invention utilized Wisconsin Package Version 9.0software from Genetics Computer Group (GCG), Madison, Wis.

SEQ ID NO:1 is the nucleotide sequence of a cloned cDNA encoding amature spinach LS.

SEQ ID NO:2 is the deduced amino acid sequence of the cloned cDNAencoding a mature spinach LS.

SEQ ID NO:3 is the nucleotide sequence of a cloned cDNA encoding amature tobacco LS.

SEQ ID NO:4 is the deduced amino acid sequence of the cloned cDNAencoding a mature tobacco LS.

SEQ ID NO:5 is the nucleotide sequence of a cloned cDNA encoding amature arabidopsis LS.

SEQ ID NO:6 is the deduced amino acid sequence of the cloned cDNAencoding a mature arabidopsis LS.

SEQ ID NO:7 is the nucleotide sequence of a cloned cDNA encoding amature spinach RS.

SEQ ID NO:8 is the deduced amino acid sequence of the cloned cDNAencoding a mature spinach RS.

SEQ ID NO:9 is the nucleotide sequence of a cloned cDNA encoding amature arabidopsis RS.

SEQ ID NO:10 is the deduced amino acid sequence of the cloned cDNAencoding a mature arabidopsis RS.

SEQ ID NO:11 is the nucleotide sequence of a cloned cDNA encoding aMagnaporthe grisea RS.

SEQ ID NO:12 is the deduced amino acid sequence of the cloned cDNAencoding Magnaporthe grisea RS.

SEQ ID NO:13 is the 5′ primer useful in the amplification of E. coli LShaving Genbank accession No. X64395.

SEQ ID NO:14 is the 3′ primer useful in the amplification of E. coli LShaving Genbank accession No. X64395.

SEQ ID NO:15 is the 5′ primer useful in the amplification of E. coli RShaving Genbank accession No. X69109.

SEQ ID NO:16 is the 3′ primer useful in the amplification of E. coli RShaving Genbank accession No. X69109.

SEQ ID NO:17 is the 5′ primer useful for the introduction of a DNAfragment that confers kanamycin resistance into the E. coli LS and RSgenes having Genbank accession Nos. X64395 and X69109, respectively, ata Not1 cleavage site.

SEQ ID NO:18 is the 3′ primer useful for the introduction of a DNAfragment that confers kanamycin resistance into E. coli LS and RS geneshaving Genbank accession Nos. X64395 and X69109, respectively, at a Not1cleavage site.

SEQ ID NO:19 is one of the PCR primers useful for the introduction of aNotI cleavage site in the middle of E. coli LS having Genbank accessionNo. X64395 (hybridizes to nt 2273-2290).

SEQ ID NO:20 is one of the PCR primers useful for the introduction of aNotI cleavage site in the middle of E. coli LS having Genbank accessionNo. X64395 (hybridizes to nt 2243-2261).

SEQ ID NO:21 is one of the PCR primers useful for the introduction of aNotI cleavage site in the middle of E. coli RS having Genbank accessionNo. X69109 (hybridizes to nt 1217-1233).

SEQ ID NO:22 is the one of the PCR primers useful for the introductionof a NotI cleavage site in the middle of E. coli RS having Genbankaccession No. X69109 (hybridizes to nt 1190-1208).

SEQ ID NO:23 is the 5′ primer useful for the removal of the transitpeptide from the cloned spinach RS precursor.

SEQ ID NO:24 is the 3′ primer useful for the removal of the transitpeptide from the cloned spinach RS precursor.

SEQ ID NO:25 is the 5′ primer useful for the removal of the transitpeptide from the cloned spinach LS precursor.

SEQ ID NO:26 is the 3′ primer useful for the removal of the transitpeptide from the cloned spinach LS precursor.

SEQ ID NO:27 is the nucleotide sequence of a cloned cDNA encoding aspinach LS precursor with its transit peptide.

SEQ ID NO:28 is the deduced amino acid sequence of the cloned cDNAencoding a spinach LS precursor with its transit peptide.

SEQ ID NO:29 is the nucleotide sequence of a cloned cDNA encoding atobacco LS precursor with its transit peptide.

SEQ ID NO:30 is the deduced amino acid sequence of the cloned cDNAencoding a tobacco LS precursor with its transit peptide.

SEQ ID NO:31 is the nucleotide sequence of a cloned cDNA encoding anarabidopsis LS precursor with its transit peptide.

SEQ ID NO:32 is the deduced amino acid sequence of the cloned cDNAencoding an arabidopsis LS precursor with its transit peptide.

SEQ ID NO:33 is the nucleotide sequence of a cloned cDNA encoding aspinach RS precursor with its transit peptide.

SEQ ID NO:34 is the deduced amino acid sequence of the cloned cDNAencoding a spinach RS precursor with its transit peptide.

SEQ ID NO:35 is the nucleotide sequence of a cloned cDNA encoding anarabidopsis RS precursor with its transit peptide.

SEQ ID NO:36 is the deduced amino acid sequence of the cloned cDNAencoding an arabidopsis RS precursor with its transit peptide.

SEQ ID NO:37 is the nucleotide sequence of a cloned cDNA encoding aMagnaporthe grisea LS.

SEQ ID NO:38 is the deduced amino acid sequence of the cloned cDNAencoding Magnaporthe grisea LS.

SEQ ID NO:39 is the highly conserved C-terminal amino acid sequencefound in plant LS proteins.

DETAILED DESCRIPTION OF THE INVENTION

Luminase synthase (LS) and riboflavin synthase (RS), the ultimate andpentultimate enzymes of the spinach riboflavin biosynthetic pathway havebeen cloned by use of function complementation of E. coli auxotrophs.

Nucleic acid fragments that respectively encode LS protein and RSprotein have been isolated from plants and fungi. LS and RS genes fromother plants and fungi can now be identified by comparison of randomcDNA sequences to the sequences provided by Applicants. The inventionincludes assays using these nucleic acid fragments to screen for cropprotection chemicals related to the enzymatic pathway and methods foraltering the levels of production of LS and RS enzymes in a host cell.

In this disclose, a number of terms and abbreviations are used. Thefollowing definitions are provided.

“Lumazine synthase” is abbreviated as LS.

“Riboflavin synthase” is abbreviated as RS.

“Flavin mononucleotide” is abbreviated as FMN.

“Flavin adenine dinucleotide” is abbreviated as FAD.

“Polymerase chain reaction” is abbreviated PCR.

“Expressed sequence tag” is abbreviated EST.

“Dimethyl sulfoxide” is abbreviated DMSO.

“6,7-Dimethyl-8-(1′-D-ribityl)lumazine” is abbreviated DMRL.

“4-Ribitylamino-5-amino-2,6-dihydroxypyrimidine” is abbreviated RAADP.

“3,4-Dihydroxybutanone 4-phosphate” is abbreviated DHBP.

“Isopropyl- 1-thio-β-D-galactopyranoside” is abbreviated IPTG.

“Sodium dodecylsulfate-polyacrylamide gel electrophoresis” isabbreviated SDS-PAGE.

“Open reading frame” is abbreviated ORF.

An “isolated nucleic acid fragment” is a polymer of RNA or DNA that issingle- or double-stranded, optionally containing synthetic, non-naturalor altered nucleotide bases. An isolated nucleic acid fragment in theform of a polymer of DNA may be comprised of one or more segments ofCDNA, genomic DNA or synthetic DNA.

“Mature” protein refers to a functional LS or RS enzyme without itstransit peptide. “Precursor” protein refers to the mature protein with anative or foreign transit peptide. The term “transit peptide” refers tothe amino terminal extension of a polypeptide, which is translated inconjunction with the polypeptide forming a precursor peptide and whichis required for its uptake by organelles such as plastids orchloroplasts.

“Auxotrophy” refers to the nutritional requirements necessary forgrowth, sporulation and crystal production of the microorganism. For thepurpose of this invention, the term “auxotroph” is defined herein tomean an organism which requires the addition of riboflavin for growth.

The terms “host cell” and “host organism” refer to a cell capable ofreceiving foreign or heterologous genes and expressing those genes toproduce an active gene product. Suitable host cells includemicroorganisms such as bacteria and fungi, as well as plant cells.

The terms “lumazine synthase” or “LS” are used interchangeably with“6,7-dimethyl-8-ribityllumazine synthase” and refer to a plant or fungalenzyme that catalyzes the condensation of 3,4-dihydroxy-2-butanone4-phosphate with 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine (RAADP)to yield orthophosphate and 6,7-dimethyl-8-(1′-D-ribityl)-lumazine(DMRL).

The terms “riboflavin synthase” or “RS” refer to a plant or fungalenzyme that catalyzes the dismutation of6,7-dimethyl-8-(1′-D-ribityl)-lumazine (DMRL) to yield riboflavin and4-ribitylamino-5-amino-2,6-dihydroxypyrimidine (RAADP).

The term “metabolic activity” refers to the normal cellular activityneeded to support growth. As used herein agents such as crop protectionchemicals that will inhibit metabolic activity will also generallyinhibit cell growth.

The term, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases result in substitutionof one or more amino acids, but do not affect the functional propertiesof the protein encoded by the DNA sequence. “Substantially similar” alsorefers to nucleic acid fragments wherein changes in one or morenucleotide bases do not affect the ability of the nucleic acid fragmentto mediate alteration of gene expression by antisense or co-suppressiontechnology. “Substantially similar” also refers to modifications of thenucleic acid fragments of the instant invention such as deletion orinsertion of one or more nucleotide bases that do not substantiallyaffect the functional properties of the resulting transcript vis-à-visthe ability to mediate alteration of gene expression by antisense orco-suppression technology or alteration of the functional properties ofthe resulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary sequences.

A “substantial portion” refers to an amino acid or nucleotide sequencewhich comprises enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to afford putative identification ofthat polypeptide or gene, either by manual evaluation of the sequence byone skilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Basic Local AlignmentSearch Tool; Altschul et al., J. Mol. Biol. 215:403-410 (1993); see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides (generally 12 bases orlonger) may be used as amplification primers in PCR in order to obtain aparticular nucleic acid fragment comprising the primers. Accordingly, a“substantial portion” of a nucleotide sequence comprises enough of thesequence to afford specific identification and/or isolation of a nucleicacid fragment comprising the sequence. The instant specification teachespartial or complete amino acid and nucleotide sequences encoding one ormore particular plant proteins. The skilled artisan, having the benefitof the sequences as reported herein, may now use all or a substantialportion of the disclosed sequences for the purpose known to thoseskilled in the art. Accordingly, the instant invention comprises thecomplete sequences as reported in the accompanying Sequence Listing, aswell as substantial portions of those sequences as defined above.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% identity with the geneto be suppressed. Moreover, alterations in a gene which result in theproduction of a chemically equivalent amino acid at a given site, but donot effect the functional properties of the encoded protein, are wellknown in the art. Thus, a codon for the amino acid alanine, ahydrophobic amino acid, may be substituted by a codon encoding anotherless hydrophobic residue, such as glycine, or a more hydrophobicresidue, such as valine, leucine, or isoleucine. Similarly, changeswhich result in substitution of one negatively charged residue foranother, such as aspartic acid for glutamic acid, or one positivelycharged residue for another, such as lysine for arginine, can also beexpected to produce a functionally equivalent product. Nucleotidechanges which result in alteration of the N-terminal and C-terminalportions of the protein molecule would also not be expected to alter theactivity of the protein. Each of the proposed modifications is wellwithin the routine skill in the art, as is determination of retention ofbiological activity of the encoded products. Moreover, the skilledartisan recognizes that substantially similar sequences encompassed bythis invention are also defined by their ability to hybridize, understringent conditions (0.1×SSC, 0.1% SDS, 65° C.), with the sequencesexemplified herein. Preferred substantially similar nucleic acidfragments of the instant invention are those nucleic acid fragmentswhose DNA sequences are 80% identical to the DNA sequence of the nucleicacid fragments reported herein. More preferred nucleic acid fragmentsare 90% identical to the DNA sequence of the nucleic acid fragmentsreported herein. Most preferred are nucleic acid fragments that are 95%identical to the DNA sequence of the nucleic acid fragments reportedherein.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include but is not limited to the GCG suite of programs (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.),BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410(1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715USA). Within the context of this application it will be understood thatwhere sequence analysis software is used for analysis, that the resultsof the analysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default vales”will mean any set of values or parameters which originally load with thesoftware when first initialized.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data PartI (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey(1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.)Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. andDevereux, J., eds.) Stockton Press, New York (1991). Preferred methodsto determine identity are designed to give the largest match between thesequences tested. Methods to determine identity and similarity arecodified in publicly available computer programs. Preferred computerprogram methods to determine identity and similarity between twosequences include, but are not limited to, the GCG Pileup program foundin the GCG program package, using the Needleman and Wunsch algorithmwith their standard default values of gap creation penalty=12 and gapextension penalty=4 (Devereux et al., Nucleic Acids Res. 12:387-395(1984)), BLASTP, BLASTN, and FASTA (Pearson et al., Proc. Natl. Acad.Sci. USA 85:2444-2448 (1988). The BLASTX program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul et al., Natl. Cent.Biotechnol. Inf., Natl. Library Med. (NCBI NLM) NIH, Bethesda, Md.20894; Altschul et al., J. Mol. Biol. 215:403-410 (1990); Altschul,Stephen F., Thomas L. Madden, Alejandro A. Schäffer, Jinghui Zhang,Zheng Zhang, Webb Miller, and David J. Lipman, “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402 (1997)). Another preferred method todetermine percent identity, is by the method of DNASTAR proteinalignment protocol using the Jotun-Hein algorithm (Hein et al., MethodsEnzymol. 183:626-645 (1990)). Default parameters for the Jotun-Heinmethod for alignments are: for multiple alignments, gap penalty=11, gaplength penalty=3; for pairwise alignments ktuple=2. As an illustration,by a polynucleotide having a nucleotide sequence having at least, forexample, 95% “identity” to a reference nucleotide sequence it isintended that the nucleotide sequence of the polynucleotide is identicalto the reference sequence except that the polynucleotide sequence mayinclude up to five point mutations per each 100 nucleotides of thereference nucleotide sequence. In other words, to obtain apolynucleotide having a nucleotide sequence at least 95% identical to areference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations of the reference sequence may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. Analogously, by a polypeptidehaving an amino acid sequence having at least, for example, 95% identityto a reference amino acid sequence is intended that the amino acidsequence of the polypeptide is identical to the reference sequenceexcept that the polypeptide sequence may include up to five amino acidalterations per each 100 amino acids of the reference amino acid. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

“Codon degeneracy” refers to redundancy in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment that encodes all or a substantialportion of the amino acid sequence encoding the LS or RS biosyntheticenzymes as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:38. The skilled artisan iswell aware of the “codon-bias” exhibited by a specific host cell inusage of nucleotide codons to specify a given amino acid. Therefore,when synthesizing a gene for improved expression in a host cell, it isdesirable to design the gene such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

The term “complementary” is used to describe the relationship betweennucleotide bases that are hybridizable to one another. Hence withrespect to DNA, adenosine is complementary to thymine and cytosine iscomplementary to guanine.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength. Hybridization and washing conditions are well known andexemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 andTable 11.1 therein (entirely incorporated herein by reference). Theconditions of temperature and ionic strength determine the “stringency”of the hybridization. For preliminary screening for homologous nucleicacids, low stringency hybridization conditions, corresponding to a Tm of55°, can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide;or 30% formamide, 5×SSC, 0.5% SDS. Moderate stringency hybridizationconditions correspond to a higher Tm, e.g., 40% formamide, with 5× or6×SSC.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (see Sambrooket al., supra, 9.50-9.51). For hybridizations with shorter nucleicacids, i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook et al., supra, 11.7-11.8). In one embodimentthe length for a hybridizable nucleic acid is at least about 10nucleotides. Preferably a minimum length for a hybridizable nucleic acidis at least about 15 nucleotides; more preferably at least about 20nucleotides; and most preferably the length is at least 30 nucleotides.Furthermore, the skilled artisan will recognize that the temperature andwash solution salt concentration may be adjusted as necessary accordingto factors such as length of the probe.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments which are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters whichcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters”. New promoters ofvarious types useful in plant cells are constantly being discovered;numerous examples may be found in the compilation by Okamuro andGoldberg, (Biochemistry of Plants 15:1-82 (1989)). It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of differentlengths may have identical promoter activity.

The “translation leader sequence” refers to a DNA sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner et al., Mol. Biotech. 3:225(1995)).

The “3′ non-coding sequences” refer to DNA sequences located downstreamof a coding sequence and include polyadenylation recognition sequencesand other sequences encoding regulatory signals capable of affectingmRNA processing or gene expression. The polyadenylation signal isusually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al., Plant Cell1:671-680 (1989).

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA” (mRNA) refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065).The complementarity of an antisense RNA may be with any part of thespecific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, introns, or the coding sequence. “Functional RNA”refers to antisense RNA, ribozyme RNA, or other RNA that is nottranslated yet has an effect on cellular processes.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020).

“Altered levels” refers to the production of gene product(s) inorganisms in amounts or proportions that differ from that of normal ornon-transformed organisms.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al., Meth. Enzymol. 143:277 (1987)) andparticle-accelerated or “gene gun” transformation technology (Klein etal., Nature, London 327:70-73 (1987); U.S. Pat. No. 4,945,050).

“Chemical compound of interest” and “test compound” refer to thematerial which is being screened in the instant assay to assess itspotential as an herbicide or fungicide crop protection chemical.

A spinach LS has been isolated and identified by comparison of randomcDNA sequences to the GenBank database using the BLAST algorithms wellknown to those skilled in the art. The nucleotide sequence of maturespinach LS cDNA is provided in SEQ ID NO:1, and the deduced amino acidsequence is provided in SEQ ID NO:2. LS genes from other plants can nowbe identified by comparison of random cDNA sequences to the spinach LSsequence provided herein.

A tobacco LS has been isolated and identified by comparison of randomcDNA sequences to the GenBank database using the BLAST algorithms wellknown to those skilled in the art. The nucleotide sequence of maturetobacco LS cDNA is provided in SEQ ID NO:3, and the deduced amino acidsequence is provided in SEQ ID NO:4. LS genes from other plants can nowbe identified by comparison of random cDNA sequences to the tobacco LSsequence provided herein.

An arabidopsis LS has been isolated and identified by comparison ofrandom cDNA sequences to the GenBank database using the BLAST algorithmswell known to those skilled in the art. The nucleotide sequence ofmature arabidopsis LS cDNA is provided in SEQ ID NO:5, and the deducedamino acid sequence is provided in SEQ ID NO:6. LS genes from otherplants can now be identified by comparison of random cDNA sequences tothe arabidopsis LS sequence provided herein.

A Magnaporthe grisea LS has been isolated and identified by comparisonof random cDNA sequences to the GenBank database using the BLASTalgorithms well known to those skilled in the art. The nucleotidesequence of Magnaporthe grisea LS cDNA is provided in SEQ ID NO:37, andthe deduced amino acid sequence is provided in SEQ ID NO:38. LS genesfrom other fungi can now be identified by comparison of random cDNAsequences to the Magnaporthe grisea LS sequence provided herein.

A spinach RS has been isolated and identified by comparison of randomcDNA sequences to the GenBank database using the BLAST algorithms wellknown to those skilled in the art. The nucleotide sequence of maturespinach RS cDNA is provided in SEQ ID NO:7, and the deduced amino acidsequence is provided in SEQ ID NO:8. RS genes from other plants can nowbe identified by comparison of random cDNA sequences to the spinach RSsequence provided herein.

An arabidopsis RS has been isolated and identified by comparison ofrandom cDNA sequences to the GenBank database using the BLAST algorithmswell known to those skilled in the art. The nucleotide sequence ofmature arabidopsis RS cDNA is provided in SEQ ID NO:9, and the deducedamino acid sequence is provided in SEQ ID NO:10. RS genes from otherplants can now be identified by comparison of random cDNA sequences tothe arabidopsis RS sequence provided herein.

A Magnaporthe grisea RS has been isolated and identified by comparisonof random cDNA sequences to the GenBank database using the BLASTalgorithms well known to those skilled in the art. The nucleotidesequence of Magnaporthe grisea RS cDNA is provided in SEQ ID NO:11, andthe deduced amino acid sequence is provided in SEQ ID NO:12. RS genesfrom other fungi can now be identified by comparison of random cDNAsequences to the Magnaporthe grisea RS sequence provided herein.

The nucleic acid fragments of the instant invention may be used toisolate cDNAs and genes encoding a homologous LS and RS from the same orother plant or fungal species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction (PCR) or ligase chain reaction).

For example, LS or RS genes, either as cDNAs or genomic DNAs, could beisolated directly by using all or a portion of the instant nucleic acidfragments as DNA hybridization probes to screen libraries from anydesired plant (or fungus) employing methodology well known to thoseskilled in the art. Specific oligonucleotide probes based upon theinstant LS or RS sequences can be designed and synthesized by methodsknown in the art (Maniatis supra). Moreover, the entire sequences can beused directly to synthesize DNA probes by methods known to the skilledartisan such as random primers, DNA labeling, nick translation, orend-labeling techniques, or RNA probes using available in vitrotranscription systems. In addition, specific primers can be designed andused to amplify a part of or full-length of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragment maybe used in PCR protocols to amplify longer nucleic acid fragmentsencoding homologous LS or RS genes from DNA or RNA. The polymerase chainreaction may also be performed on a library of cloned nucleic acidfragments wherein the sequence of one primer is derived from the instantnucleic acid fragments, and the sequence of the other primer takesadvantage of the presence of the polyadenylic acid tracts to the 3′ endof the mRNA precursor encoding plant LS or RS. Alternatively, the secondprimer sequence may be based upon sequences derived from the cloningvector. For example, the skilled artisan can follow the RACE protocol(Frohman et al., Proc. Natl. Acad. Sci., USA 85:8998 (1988)) to generatecDNAs by using PCR to amplify copies of the region between a singlepoint in the transcript and the 3′ or 5′ end. Primers oriented in the 3′and 5′ directions can be designed from the instant sequences. Usingcommercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or5′ cDNA fragments can be isolated (Ohara et al., Proc. Natl. Acad. Sci.,USA 86:5673 (1989); Loh et al., Science 243:217 (1989)). Productsgenerated by the 3′ and 5′ RACE procedures can be combined to generatefull-length cDNAs (Frohman et al., Techniques 1:165 (1989)).

Finally, availability of the instant nucleotide and deduced amino acidsequences facilitates immunological screening cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner et al., Adv.Immunol. 36:1 (1984); Maniatis).

The nucleic acid fragments of the instant invention may also be used tocreate transgenic plants in which the instant LS or RS proteins arepresent at higher or lower levels than normal. Such manipulations wouldconceivably alter the intracellular levels of riboflavin, hence theessential cofactors FAD and FMN, producing novel phenotypes of potentialcommercial value. Alternatively, in some applications, it might bedesirable to express the instant LS or RS proteins in specific planttissues and/or cell types, or during developmental stages in which theywould normally not be encountered.

Overexpression of the instant LS or RS may be accomplished by firstconstructing a chimeric gene in which the LS or RS coding region isoperably linked to a promoter capable of directing expression of a genein the desired tissues at the desired stage of development. For reasonsof convenience, the chimeric gene may comprise promoter sequences andtranslation leader sequences derived from the same genes. 3′ Non-codingsequences encoding transcription termination signals must also beprovided. The instant chimeric genes may also comprise one or moreintrons in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric genes can then beconstructed. The choice of a plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al., EMBO J.4:2411-2418 (1985); De Almeida et al., Mol. Gen. Genetics 218:78-86(1989)), and thus that multiple events must be screened in order toobtain lines displaying the desired expression level and pattern. Suchscreening may be accomplished by Southern analysis of DNA, Northernanalysis of mRNA expression, Western analysis of protein expression, orphenotypic analysis.

For some applications it may be useful to direct the LS or RS proteinsto different cellular compartments or to facilitate their secretion fromthe cell. It is thus envisioned that the chimeric genes described abovemay be further modified by the addition of appropriate intracellular orextracellular targeting sequences to their coding regions. These includechloroplast transit peptides (Keegstra et al., Cell 56:247-253 (1989),signal sequences that direct proteins to the endoplasmic reticulum(Chrispeels et al., Ann. Rev. Plant Phys. Plant Mol. 42:21-53 (1991),and nuclear localization signals (Raikhel et al., Plant Phys.100:1627-1632 (1992). While the references cited give examples of eachof these, the list is not exhaustive and more targeting signals ofutility may be discovered in the future. As described below, it isdemonstrated in the present invention that plant LS and RS are bothsynthesized as nuclear-encoded precursor proteins with chloroplasttargeting sequences at their N-termini. Thus, these proteins appear tobe good candidates for targeting to other cellular compartments.Alternatively, by simply removing their chloroplast transit peptides, itshould be possible to express LS or RS exclusively in the plant cytosol.

It may also be desirable to reduce or eliminate expression of the LS orRS genes in plants for some applications. In order to accomplish this,chimeric genes designed for antisense or co-suppression of LS or RS canbe constructed by linking the genes or gene fragments encoding parts ofthese enzymes to plant promter sequences. Thus, chimeric genes designedto express antisense RNA for all or part of LS or RS can be constructedby linking the LS or RS genes or gene fragments in reverse orientationto plant promoter sequences. The co-suppression or antisense chimericgene constructs could then be introduced into plants via well knowntransformation protocols to reduce or eliminate the endogenousexpression of LS or RS gene products.

The LS or RS protein produced in heterologous host cells, particularlyin the cells of microbial hosts, can be used to prepare antibodies tothe enzymes by methods well known to those skilled in the art. Theantibodies would be useful for detecting the instant LS or RS protein insitu in cells or in vitro in cell extracts. Preferred heterologous hostcells for production of the instant LS or RS protein are microbialhosts. Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct chimeric genes for production of the instant LS orRS. These chimeric genes could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe instant LS or RS protein.

Microbial host cells suitable for the expression of the instant LS andRS enzymes include any cell capable of expression of the chimeric genesencoding these enzymes. Such cells will include both bacteria and fungiincluding, for example, the yeasts (e.g., Aspergillus, Saccharomyces,Pichia, Candida, and Hansenula), members of the genus Bacillus as wellas the enteric bacteria (e.g., Escherichia, Salmonella, and Shigella).Methods for the transformation of such hosts and the expression offoreign proteins are well known in the art and examples of suitableprotocols may be found in Manual of Methods for General Bacteriology(Gerhardt et al., eds., American Society for Microbiology, Washington,D.C. (1994) or in Brock, T. D., Biotechnology: A Textbook of IndustrialMicrobiology, Second Edition, Sinauer Associates, Inc., Sunderland,Mass. (1989)).

Vectors or cassettes useful for the transformation of suitable microbialhost cells are well known in the art. Typically the vector or cassettecontains sequences directing transcription and translation of therelevant gene, a selectable marker, and sequences allowing autonomousreplication or chromosomal integration. Suitable vectors comprise aregion 5′ of the gene which harbors transcriptional initiation controlsand a region 3′ of the DNA fragment which controls transcriptionaltermination. It is most preferred when both control regions are derivedfrom genes homologous to the transformed host cell although it is to beunderstood that such control regions need not be derived from the genesnative to the specific species chosen as a production host.

Initiation control regions or promoters, which are useful to driveexpression of the genes encoding the RS or LS enzymes in the desiredhost cell are numerous and familiar to those skilled in the art.Virtually any promoter capable of driving these genes is suitable forthe present invention including but not limited to CYC1, HIS3, GAL1,GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (usefulfor expression in Saccharomyces); AOX1 (useful for expression inPichia); and lac, trp, 1P_(L), 1P_(R), T7, tac, and trc (useful forexpression in E. coli). Termination control regions may also be derivedfrom various genes native to the preferred hosts. Optionally, atermination site may be unnecessary, however, it is most preferred ifincluded.

The instant LS and RS proteins can be used as tools to facilitate thedesign and/or identification of specific chemical agents that mightprove useful as herbicides or fungicides. This could be achieved eitherthrough the rational design and synthesis of potent enzyme inhibitorsthat result from structural and/or mechanistic information that isderived from the purified instant plant proteins, or through random invitro screening of chemical libraries. LS and RS catalyze the last twosteps of riboflavin biosynthesis in plants and microorganism, and arerequired for the production of FAD and FMN, essential prosthetic groupsfor a number of important redox enzymes. Consequently, it is anticipatedthat significant in vivo inhibition of any of the LS or RS proteinsdescribed herein will severely cripple cellular metabolism and likelyresult in plant or fungal death.

All or a portion of the nucleic acid fragments of the instant inventionmay also be used as probes for genetically and physically mapping thegenes that they are a part of, and as markers for traits linked toexpression of the instant LS or RS. Such information may be useful inplant breeding in order to develop lines with desired phenotypes.

For example, the instant nucleic acid fragments may be used asrestriction fragment length polymorphism (RFLP) markers. Southern blots(Maniatis) of restriction-digested plant genomic DNA may be probed withthe nucleic acid fragments of the instant invention. The resultingbanding patterns may then be subjected to genetic analyses usingcomputer programs such as MapMaker (Lander et at., Genomics 1:174-181(1987)) in order to construct a genetic map. In addition, the nucleicacid fragments of the instant invention may be used to probe Southernblots containing restriction endonuclease-treated genomic DNAs of a setof individuals representing parent and progeny of a defined geneticcross. Segregation of the DNA polymorphisms is noted and used tocalculate the position of the instant nucleic acid sequence in thegenetic map previously obtained using this population (Botstein et al.,Am. J. Hum. Genet. 32:314-331 (1980)).

The production and use of plant gene-derived probes for use in geneticmapping is described by Bernatzky and Tanksley (Plant Mol. Biol.Reporter 4:37-41 (1986)). Numerous publications describe genetic mappingof specific cDNA clones using the methodology outlined above orvariations thereof. For example, F2 intercross populations, backcrosspopulations, randomly mated populations, near isogenic lines, and othersets of individuals may be used for mapping. Such methodologies are wellknown to those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al., Nonmammalian Genomic Analysis: APractical Guide, pp. 319-346, Academic Press (1996), and referencescited therein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequence may be used in direct fluorescence in situhybridization (FISH) mapping. Although current methods of FISH mappingfavor use of large clones (several to several hundred kb), improvementsin sensitivity may allow performance of FISH mapping using shorterprobes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification, polymorphismof PCR-amplified fragments (CAPS), allele-specific ligation, nucleotideextension reactions, Radiation Hybrid Mapping and Happy Mapping. Forthese methods, the sequence of a nucleic acid fragment is used to designand produce primer pairs for use in the amplification reaction or inprimer extension reactions. The design of such primers is well known tothose skilled in the art. In methods employing PCR-based geneticmapping, it may be necessary to identify DNA sequence differencesbetween the parents of the mapping cross in the region corresponding tothe instant nucleic acid sequences. This, however, is generally notnecessary for mapping methods. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usage and conditions.

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook, J., Fritsch, E. F.and Maniatis, T. Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, 1989; and by T. J. Silhavy,M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, ColdSpring Harbor Laboratory Press, Cold Spring, N.Y. (1984) and by Ausubel,F. M. et al., Current Protocols in Molecular Biology, pub. by GreenePublishing Assoc. and Wiley-Interscience (1987).

Manipulations of genetic sequences were accomplished using the suite ofprograms available from the Genetics Computer Group Inc. (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.).Where the GCG program “Pileup” was used the gap creation default valueof 12, and the gap extension default value of 4 were used. Where the CGC“Gap” or “Bestfit” programs were used the default gap creation penaltyof 50 and the default gap extension penatly of 3 were used. In any casewhere GCG program parameters were not prompted for, in these or anyother GCG program, default values were used.

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “h” means hour(s), “d” means day(s), “ml” meansmicroliter, “mL” means milliliters, “L” means liters, “mM” meansmillimolar, “M” means molar, “mmol” means millimole(s).

EXAMPLE 1 PCR-Cloning of E. Coli LS and RS

Gene specific PCR primers were used to amplify the E. coli LS and RSgenes from genomic DNA, while adding unique restriction sites to theirflanking regions for subsequent ligation into high copy number plasmids.The primers used for this purpose were based on the published DNAsequences of the E. coli LS and RS genes (GenBank accession numbersX64395 and X69109, respectively) and consisted of the followingnucleotides:

Primer 1-(SEQ ID NO:13):

5′-CGA AGG AAG Acc atg gCC ATT ATT GAA GCT AAC GTT GC-3′

Primer 2-(SEQ ID NO:14):

5′-ATC TTA CTg tcg acT TCA GGC CTT GAT GGC TTT C-3′

Primer 3-(SEQ ID NO:15):

5′-ACT CAT TTA cca tgg CTA CGG GGA TTG TAC AGG GC-3′

Primer4-(SEQ ID NO:16):

5′-ATC TTA CTg tcg acT TCA GGC TTC TGT GCC TGG TT-3′

The underlined bases hybridize to the target genes, while lower caseletters indicate the restriction sites (NcoI or SalI) that were added tothe ends of the PCR primers.

Ampliflication of the LS gene was achieved using Primers 1 and 2, andgenomic DNA from E. coli strain W3110 (Campbell et al., Proc. Natl.Acad. Sci. 75:2276-2284 (1978)). Primer 1 hybridizes at the start of thegene and introduces a NcoI site at the protein's initiation codon, whilePrimer 2 hydridizes at the opposite end and provides a SalI site justpast the termination codon. The 100-μl PCR reactions contained ˜100 ngof genomic DNA and both primers at a final concentration of 0.5μM. Theother reaction components were provided by the GeneAmp PCR Reagent Kit(Perkin Elmer), according to the manufacturer's protocol. Amplificationwas carried out in a DNA Thermocycler 480 (Perkin Elmer) for 28 cycles,each comprising 1 min at 94° C., 2 min at 53° C., and 2 min at 72° C.Following the last cycle, there was a 7-min extension period at 72° C.The PCR product was cut with NcoI and SalI, and ligated into similarlydigested pGEM-5Zf (+) (Promega, Madison, Wis.). The latter was chosen asa suitable cloning vector since it lacks a NotI cleavage site afterdouble-digestion with NcoI and SalI (see below). The ligation reactionmixture was used to transform E. coli DH5α competant cells (GibcoBRL),and transformants were selected on LB media supplemented with 100 μg/mLampicillin.

The E. coli RS gene was amplified from genomic DNA in a similar mannerusing Primers 3 and 4. The former introduces a NcoI site at theprotein's initiation codon, while the latter provides a SalI site justafter the stop codon. Subsequent steps, including ligation of the PCRproduct into pGEM-5Zf (+) and transformation of DH5α with the resultingconstruct were exactly as described above.

Plasmids harboring the cloned E. coli LS and RS genes were identified byrestriction digestion analysis. Plasmid DNA was isolated from a numberof ampicillin-resistant colonies using the Wizard DNA PurificationSystem (Promega, Madison, Wis.) and subjected to cleavage with NcoI andSalI. The samples were analyzed by agarose gel electrophoresis, and arepresentative plasmid for each gene, yielding inserts of the correctsize, was sequenced completely to verify the absence of PCR errors.Apart from those nucleotides at the 5′ and 3′ ends that wereintentionally altered for cloning purposes, the amplified E. coli LS andRS gene sequences were identical to those reported in the literature.

EXAMPLE 2 Insertional Inactivation of the E. Coli LS and RS Genes

In order to create bacterial auxotrophs lacking the ability tosynthesize riboflavin, the cloned E. coli LS and RS genes were renderednonfunctional through insertional inactivation. Briefly, a unique NotIsite was introduced in the middle of the coding region of each of thetarget genes, and a DNA fragment that confers kanamycin resistance wasligated into the engineered sites. The latter was provided by thecommerically available Kan^(r) GenBlock cartridge (Pharmacia), that wasmodified through PCR to add NotI cleavage sites at both of its ends.This modification was accomplished using Primers 5 and 6 in a standardPCR reaction; the underlined portions hybridize to the Kan^(r) GenBlock,and lower case letters indicate the NotI cleavage sites.

Primer 5-(SEQ ID NO:17):

5′-AAC TAG ATC Agc ggc cgc AGC CAC GTT GTG TCT CAA A-3′

Primer 6-(SEQ ID NO:18):

5′-GAC AAA CAT Agc ggc cgc TGA GGT CTG CCT CGT GAA-3′

Following amplification, the modified Kan^(r) GenBlock was cleaved withNotI, and the resulting fragment was purified by agarose gelelectrophoresis.

PCR primers were also used to introduce a unique NotI cleavage site inthe middle of the two target genes. This was accomplished through anapplication of the “inverse PCR” technique that is fully described byOchman, et al. in PCR Protocols: A Guide to Methods and Applications,(Innis et al., eds.) pp. 219-227, Academic Press, San Diego, Calif.,(1990). The targets for inverse PCR are usually double-stranded circularDNA molecules. However, in contrast to other PCR applications, the twoprimers are oriented away from each other such that their 3′ ends areextended in opposite directions around the entire circular template. Ifthe primers are designed to hybridize immediately adjacent to eachother, a linear DNA fragment is produced that includes the entire vectorsequence and has as its starting and stopping points the original primerbinding sites. The net result is analogous to linearizing a circularplasmid at a specified location. By attaching appropriate nucleotidesequences to the nonhybridizing 5′ ends of both PCR primers, it istherefore possible to introduce a unique restriction site at any desiredlocation within a circular template.

Primers 7 and 8 (which hybridize to nt 2273-2290 and nt 2243-2261 of theDNA sequence in GenBank accession number X64395, respectively) weredesigned to introduce a NotI cleavage site in the middle of the E. coliLS gene; the nucleotides that hybridize to the target gene areunderlined, and NotI cleavage sites are indicated in lower case letters.

Primer 7-(SEQ ID NO:19):

5′-AAC TAG ATC Agc ggc cgc GGT ACG GTT ATT CGT GGT-3′

Primer 8-LS (SEQ ID NO:20):

5′-GAC AAA CAT Agc ggc cgc GTC GTA TTT ACC GGT-3′

Primers 9 and 10 (which hybridize to nt 1217-1233 and nt 1190-1208 ofGenBank accession number X69 109, respectively) were used to introduce aNotI cleavage site in the middle of the E. coli RS gene.

Primer 9-(SEQ ID NO:21):

5′-AAC TAG ATC Agc ggc cgc ACC ACT GCT GAA GTG GC-3′

Primer 10-RS (SEQ ID NO:22):

5′-GAC AAA CAT Agc ggc cgc GAC CTG ACA TTA AGT GTC C-3′

The circular templates for inverse PCR were the pGEM-5Zf (+) constructscontaining the E. coli LS and RS genes. The 100-μl PCR reactionscontained 0.5 ng of plasmid DNA and each of the appropriate primers at afinal concentration of 0.5μM. Amplification was carried out in a DNAThermocycler 480 (Perkin Elmer) for 30 cycles, each comprising 50 sec at94° C., 1 min at 55° C., and 3 min at 72° C. The PCR products werecleaved with NotI and the resulting fragments were purified by agarosegel electrophoresis; the excised bands were of the expected size. Next,the purified fragments were recircularized with T4 DNA ligase (Novagen)to regenerate functional plasmids, and aliquots of the ligation reactionmixtures were used to transform E. coli DH5α competent cells (GibcoBRL).Growth was selected for on LB media containing ampicillin (100 μg/mL),and plasmid DNA was isolated from a number of transformants forrestriction digestion analysis with NotI, SalI, and NcoI. For each ofthe target genes, a representative plasmid yielding the correct cleavagepatterns with these enzymes was selected for further manipulation.

To insert the kanamycin resistance gene, the two plasmid constructsdescribed above were cleaved with NotI and purified by agarose gelelectrophoresis. Each of the fragments was then individually incubatedwith a 4-fold molar excess of the modified Kan^(r) GenBlock cartridge,and subjected to a standard ligation reaction in the presence of T4 DNAligase (Novagen). Aliquots of the ligation reaction mixtures were usedto transform E. coli DH5α competant cells (GibcoBRL), and growth wasselected for on LB plates containing kanamycin (30 μg/mL) and ampicillin(100 μg/mL). Plasmids harboring the disrupted E. coli LS and RS geneswere identified by restriction digestion analysis. The plasmids werecleaved with NcoI and SalI, and were then subjected to agarose gelelectrophoresis to check for the presence of the inserted kanamycinresistance gene. Representative plasmids, yielding fragments of thecorrect size, were selected for further manipulation. DNA sequenceanalysis of these plasmids confirmed that the kanamycin resistance genehad been inserted at the correct location in both target genes.

EXAMPLE 3 Generation of E. Coli LS and RS Auxotrophs

The insertionally inactivated E. coli LS and RS genes were liberatedfrom the plasmid constructs described above using NcoI and SalI andpurified by agarose gel electrophoresis. Each of the fragments was thenindividually introduced into E. coli strain ATCC 47002 (fully describedin Balbas et al., Gene 136:211-213 (1993), and isogenic with JC7623(described by Bachmann, B., in E. coli and Salmonella typhimurium:Cellular and Molecular Biology (Niedhardt et al., eds.) p. 2466,American Society of Microbiology, Washington, D.C. (1987)) byelectroporatation using a BTX Transfector 100 (Biotechnologies andExperimental Research Inc.) according to the manufacturer's protocol.The choice of this strain as the initial recipient for gene replacementwas based on its well established hyper-rec phenotype and relatedability to undergo high frequency double-crossover homologousrecombination (Wyman et al., Proc. Nat. Acad. Sci. USA 82:2880-2884(1985); Balbas et al., Gene 136:211-213 (1993); Balbas et al., Gene172:65-69 (1996)). Thus, it was anticipated that the insertionallyinactivated E. coli LS and RS genes would efficiently replace theirfunctional chromosomal counterparts in ATCC 47002 under kanamycinselection.

Following electroporation, the transformed cells were resuspended in 1.0mL of S.O.C. media (GibcoBRL) that was supplemented with riboflavin (400μg/mL), and incubated for 1 h at 37° C. Kanamycin resistance was thenselected for on LB plates at 37° C. that contained both riboflavin(400μg/mL) and kanamycin (30 μg/mL); colonies appeared 24-48 h later.Phenotypic detection of the correct chromosomal integration event wasaccomplished through replica-plating experiments. Riboflavin auxotrophsresulting from double-crossover homologous recombination of either ofthe disrupted target genes would be expected to be resistant tokanamycin, sensitive to ampicillin, and to exhibit growth only in thepresence of added riboflavin. Representative bacterial coloniesexhibiting this phenotype were selected for further study.

While ATCC 47002 is an excellent strain for creating E. coli“knockouts”, its multiple mutations in the recBCD loci render itincapable of propagating ColE1-type plasmids (Balbas et al., Gene172:65-69 (1996)). Consequently, the riboflavin auxotrophs describedabove are not suitable for screening plasmid cDNA libraries byfunctional complementation. In order to achieve this goal it wastherefore necessary to move the insertionally inactivated LS and RSgenes from the chromosome of ATCC 47002 to a suitable wildtypebackground. This manipulation was accomplished through generalized phagetransduction using P1_(vir) and standard methodologies as fullydescribed by Miller, J. H., in Experiments in Molecular Genetics, pp.201-205, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,(1972). E. coli W3110 (Campbell et al., Proc. Nat. Acad. Sci.75:2276-2284 (1978)) was selected as the recipient strain for theinsertionally inactivated LS and RS genes. Following phage transduction,bacterial growth was selected for on LB media that was supplemented withkanamycin (35 μg/mL) and riboflavin (400 μg/mL). Stable transductantsharboring the disrupted E. coli LS or RS genes were then identifiedthrough replica-plating experiments analogous to those described abovefor ATCC 47002. Thus, individual colonies were patched onto platescontaining LB media, sodium citrate (7.5 mM), magnesium sulfate (1.5mM), and kanamyacin (35μg/mL), with or without riboflavin (400 μg/mL).The LS and RS riboflavin auxotrophs that were selected for further studyand subsequent complementation cloning (see below) were only able togrow in the presence of added riboflavin, and were not resistant toampicillin (100 μg/mL) or streptomycin (25 μg/mL); sensitivity tostreptomycin is characteristic of W3110, but not of ATCC 47002.

EXAMPLE 4 Cloning of Spinach LS and RS Genes Through FunctionalComplementation

A spinach (Spinacea oleracea) cDNA expression library in Lambda Zap IIwas obtained from Stratagene (La Jolla, Calif.), and subjected to massexcision according to the manufacturer's protocol. Upon excision, theliberated cDNA inserts are contained in the plasmid vector pBluscriptwhich confers resistance to amipicillin, and allows their expression inE. coli upon induction with isopropyl-1-thio-β-D-galactopyranoside(IPTG). The resulting mixture of excised plasmids was thenelectroporated into the E. coli LS and RS riboflavin auxotrophs (bothderivatives of W3110) using a BTX Transfector 1 00 (Biotechnologies andExperimental Research Inc.) and the manufacturer's conditions. Thetransformed cells were selected for growth in the absence of addedriboflavin, on plates that contained B agar (LB media containing sodiumcitrate (7.5 mM), magnesium sulfate (1.5 mM), kanamyacin (35μg/mL),ampicillin (100 μg/mL), and IPTG (0.6 mM)). Following a 48-hr incubationperiod at 37° C., bacterial growth was observed andriboflavin-independant colonies were recovered at frequencies of about4.2×10⁻⁶ and about 3.1×10⁻⁷, for the E. coli LS and RS auxotrophs,respectively. For each of the target genes, plasmid DNA was isolatedfrom a representative colony and subjected to further analysis; both ofthe selected plasmids were capable of transforming the appropiate E.coli auxotroph to riboflavin prototrophy at high frequency. The cDNAinserts contained in these plasmids were then sequenced completely on anABI 377 automated sequencer (Applied Biosystems), using fluorescentdideoxy terminators and custom-designed primers.

The approximately 1.2 kbp spinach cDNA insert that rescued the E. coliRS auxotroph clearly encodes a riboflavin synthase. The nucleotidesequence of the open reading frame (ORF) for this protein and itspredicted primary amino acid sequence are set forth in SEQ ID NO:33 andSEQ ID NO:34, respectively. While there are unmistakable similaritiesbetween the cloned plant protein and known microbial RS homologs,including those from E. coli, B. subtilis, P. leiognathi, P. phosphoreumand S. cerevisiae (GenBank accession numbers, X69109, X51510, M90094,L1139land Z21621, respectively), there are also some significantdifferences. The most obvious one is that the spinach RS is a muchlarger protein. In comparison to its counterparts in microorganisms, itpossesses an additional 69 amino acids at its N-terminus (FIG. 1). ThisN-terminal polypeptide extension is relatively basic, rich in Ser andThr residues, and as such, resembles a chloroplast transit peptide(Gavel et al., FEBS Lett. 261:455-458 (1990)). This observationsuggested that the spinach RS is synthesized as a nuclear-encodedprecursor protein, and is subsequently targeted to chloroplasts where itis proteolytically processed to its mature form. Indeed, based on thesequence alignments shown in FIG. 1 (generated with the GeneticsComputer Group Pileup program), the predicted cleavage-site formaturation occurs between amino acid residues 69 and 70 of the spinachRS precursor, giving rise to a mature polypeptide with a molecular massof 22.8 kDa (SEQ ID NO:8). That this assignment is correct is stronglysupported by the fact that all known microbial RS homologs start withthe pentapeptide motif Met-Phe-Thr-Gly-Ile, which is apparently criticalfor function (Santos et al., J. Biol. Chem. 270:437-444 (1995)).

The notion that mature spinach RS is localized in chloroplasts is alsosupported by experimental evidence. Thus, antibodies directed againstthe purified recombinant protein (See below) specifically interact witha polypeptide of the expected size when spinach chloroplast extracts aresubjected to SDS-PAGE and Western analysis. Other experiments haveclearly demonstrated that the spinach RS precursor is imported intoplastids where it is proeolytically cleaved to its mature form. In thesestudies, the full-length spinach RS precursor was labeled with[S³⁵]-methionine through in vitro transcription/translation of thecloned gene, and subjected to in vitro protein import assays (Cline etal., J. Biol. Chem. 260, 3691-3696 (1988): Viitanen et al., J. Biol.Chem. 263, 15000-15007 (1988)) using intact isolated spinachchloroplasts.

Of the various microbial homologs that are shown in FIG. 1, the maturespinach RS (SEQ ID NO:8) shows the greatest similarity to the yeastprotein at the primary amino acid sequence level (e.g., approximately47% identity). In contrast, the mature spinach RS is approximately only35%, 42%, 34%, and 40% identical to the corresponding proteins from E.coli, B. subtilis, P. leiognathi, and P. phosphoreum, respectively.Taking into account the N-terminal chloroplast targeting sequence thatis unique to the plant protein the evolutionary divergence is evengreater.

Similar observations were made with the spinach cDNA insert that wascapable of restoring riboflavin prototrophy to the E. coli LS auxotroph.In this case, the approximately 1.3 kbp DNA fragment of the rescuingplasmid contained an ORF that encoded a much larger than normal LShomolog; the DNA sequence and predicted amino acid sequence of thelatter are shown in SEQ ID NO:27 and SEQ ID NO:28, respectively.Analogous to the situation with the spinach RS precursor, the additionallength of the cloned spinach LS (relative to its homologs inmicroorganisms, including yeast) is entirely attributable to anN-terminal chloroplast transit peptide-like extension (FIG. 2). Thus, atleast the last two steps of higher plant riboflavin biosynthesis takeplace in chloroplasts. Although immunolocalization and chloroplastprotein import experiments (similar to those described above for spinachRS) have demonstrated that the spinach LS precursor is also targeted toplastids, it is more difficult in this case to predict with certaintythe exact start of the mature protein. Even amongst known microbial LShomologs it is apparent that the first 15-20 N terminal amino acidresidues are poorly conserved. Nevertheless, from the sequencealignments in FIG. 2, it is likely that the critical cleavage event formaturation occurs between Ala66 and Val67 of the spinach LS precursor,to yield a polypeptide with a predicted molecular mass of approximately16.5 kDa. While this notion remains to be determined experimentally, thepredicated amino acid sequence of the mature spinach LS based on thisassignment is given in SEQ ID NO:2. Note that even without itschloroplast targeting sequence, the mature spinach LS is only 49%, 47%,44%, 43% and 29% identical to its counterparts in E. coli, A.pleuropneumoniae, B. substilis, P. phosphoreum and S. cerevisiae,respectively (e.g., the other proteins shown in FIG. 2).

EXAMPLE 5 Expression of Mature Spinach LS and RS in E. Coli

The chloroplast targeting sequence of the cloned spinach RS precursor,identified in Example 4, was removed through a standard PCR reactionusing primers 11 (SEQ ID NO:23) and 12 (SEQ ID NO:24). Primer 11 (5′-CTACTC ATT TCA TAT GTT CAC TGG CAT TGT TGA A-3′) (SEQ ID NO:23) hydridizesto nt 208-228 of the spinach RS precursor (SEQ ID NO:33). It wasdesigned to initiate protein synthesis in E. coli at the predicted startof the mature protein (SEQ ID NO:8), and incorporates a unique NdeI siteupstream from the initiator Met residue for cloning purposes. Primer 12(5′-CAT CTT ACT GGA TCC ACT ATG TGA ATT TGG TAG GAT C-3′) (SEQ ID NO:24)hybridizes at the other end of the ORF to nt 820-840 and introduces aunique BamHI site just past the protein's stop codon. The target for PCRamplification was the purified plasmid containing the cDNA insert forthe spinach RS precursor. The predicted PCR product encodes thefull-length mature spinach RS without any modifications (SEQ ID NO:8).

A similar strategy was employed to remove the transit peptide from thecloned spinach LS precursor for expression in E. coli. However, in thiscase, it was also necessary to provide the truncated plant protein withan initiator Met residue at the predicted transit peptide cleavage-sitesince a naturally occurring one was lacking. This was accomplished usingprimers 13 (SEQ ID NO:25) and 14 (SEQ ID NO:26) in a standard PCRreaction with purified plasmid containing the cDNA insert for thespinach LS precursor. Primer 13 (5′-CTA CTC ATT TCA TAT GAA CGA GCT TGAAGG TTA TGT CAC-3′) (SEQ ID NO:25) hybridizes to nt 205-224 of thespinach LS precursor (SEQ ID NO:27), and introduces an initiator Metresidue at the position currently occupied by Val67 (SEQ ID NO:28), thepredicted start of the mature protein (SEQ ID NO:2). This primer alsoprovides a unique NdeI site at the introduced initiator Met residue andchanges the second amino acid from Arg to Asn. It was reasoned thatthese changes would not compromise enzyme acitivity, and might actuallyimprove bacterial expression of the modified plant protein, since boththe E. coli and B. subtilis LS homologs start with the dipeptidesequence, Met-Asn. Primer 14 (5′-CAT CTT ACT GGA TCC ATC AGG CCT TCA AATGAT GTT CG-3′) (SEQ ID NO:26) hybridizes at the other end of the spinachLS precursor to nt 648-667, and provides a unique BamHI site just pastthe termination codon. Thus, with the exception of the first two aminoacids, the PCR fragment generated with primers 13 and 14 will encode apolypeptide with the same primary amino acid sequence as that shown inSEQ ID NO:2.

Following amplification of the two target genes, the PCR fragments werecleaved with NdeI and BamHI, and were individually ligated intosimiliarly digested pET-24a (+) (Novagen). The latter is a high-level E.coli expression vector that is under the control of the T7 promoter.Aliquots of the ligation reaction mixtures were then used to transformE. coli BL21(DE3) using a BTX Transfector 100 (Biotechnologies andExperimental Research Inc.) according to the manufacturer's protocol.The transformed cells were plated on LB media containing kanamyacin (50μg/mL) and incubated at 37° C. to obtain single colonies. Clonesharboring plasmids with the correct inserts were identified through PCRreactions using individual resuspended colonies and the appropriateprimer pairs (i.e., primers 11 and 12 for the mature spinach RSconstruct and primers 13 and 14 for the mature spinach LS construct).Following this procedure, a representative clone for each of the targetgenes was selected for further manipulation and these two strains wereused for the production of recombinant proteins as described below.Plasmid DNA from these strains was sequenced completely to check for PCRerrors, and in both cases, none were found.

For overexpression of the mature spinach LS and RS proteins, theBL21(DE3) strains described above were grown in LB media containingkanamycin (50μg/mL) at 37° C. The cells were induced with IPTG (1 mM) atan A_(600 nm) of about 1.0, and were harvested 3 h later bycentrifugation. Both plant proteins were well expressed in the bacterialhost at levels exceeding 15% of the total soluble protein. Subsequentmanipulations were at 0-4° C. Cell pellets containing recombinantspinach RS were resuspended in 2.5 vol of 0.1 M potassium phosphate (pH7.2), 10 mM sodium sulfite, 10 mM EDTA, and passed twice through aFrench pressure cell at 20,000 psi. Debris was removed by centrifugation(10^(5 l ×g,) 1 h), and the cell-free extract, containing 44 mg ofprotein/mL, was supplemented with glycerol (5%) and stored at −80° C.for subsequent use. Protein concentrations were determined by the methodof Lowry et al. (Lowry et al., J. Biol Chem. 193:265-275 (1951)), usingBSA as a standard. Cell pellets containing recombinant spinach LS weredisrupted in an identical manner, but the buffer used for cellresuspension was 100 mM Tris-HCl (pH 7.7), 5 mM MgSO₄, 0.03 mg/mL DNAseI (Sigma), 0.5 mM phenylmethylsulfonyl flouride, 1 mM dithiothreitol andthe protein concentration of the cell-free extract was 39 mg/mL.SDS-PAGE analysis of the cell-free extracts revealed that both plantproteins were well expressed in the bacterial host, at levels exceeding20% of the total soluble protein.

EXAMPLE 6 Purification of Recombinant Mature Spinach RS

An aliquot (0.5 mL) of E. coli cell-free extract containing therecombinant spinach RS was rapidly thawed to room temperature, diluted1:1 with deionized water, and filtered through a 0.2 μMm Acrodisc filter(Gelman Sciences, Cat. No. 4192). The entire sample was then applied toa Mono Q HR 5/5 column (Pharmacia Biotech Inc), preequilibrated at 25°C. with Buffer Q (50 mM Tris-HCl, pH 7.7, 10 mM sodium sulfite, 1 mMEDTA). The column was developed at 1.0 mL/min with a linear gradient (30mL) of 0-1.0 M NaCl (in Buffer Q), and 1-mL fractions were collected.The position in the gradient where spinach RS elutes was determined bySDS-PAGE (Laemmli U., Nature 227:680-685 (1970)) using 15% gels andCoomassie Blue staining. Subsequently, column fractions eluting between0.167-0.233 M NaCl were pooled and concentrated in a Centricon-10(Amicon Inc.) at 4° C. to a final volume of 450 μL. In the next step,half of this material was applied to a 7.5×600 mm TSK G3000SW gelfiltration column (TOSOH Corp.) that was preequilibrated with Buffer Qcontaining 0.3 M NaCl. The column was developed at a flow rate of 1.0mL/min (25° C.), and highly purified spinach RS eluted between 15.2-16.2min. The latter was kept on ice while the remaining half of the samplewas processed in an identical manner. The peak fractions from the twogel filtration columns were pooled, supplemented with glycerol (5%),concentrated to 6.6 mg of protein/mL, and stored at −80° C. forsubsequent use. The yield of purified protein was 2.9 mg, correspondingto 13% of the total protein present in the cell-free extract. Visualinspection of overloaded Coomassie-stained gels suggested the finalpreparation was >95% pure.

Edman degradation of the purified recombinant spinach RS revealed thatits first 21 amino acids are identical to those of the protein shown inSEQ ID NO:8, in accord with the PCR strategy that was employed in itsconstruction. This further indicates that the recombinant protein'sN-terminus remained intact during the purification procedure. Asdetermined by electrospray ionization mass spectrometry, the protomermolecular mass of the purified recombinant spinach RS was 22808.3daltons, a value that is in excellent agreement with that predicted fromits DNA sequence (22807.26 daltons). Similar to the E. coli (Bacher etal., J. Biol Chem. 255:632-637 (1980)) and yeast (Santos et al., J.Biol. Chem. 270:437-444 (1995)) RS homologs, both of which are trimersin the native state, the recombinant spinach RS eluted during analyticalgel filtration with an apparent molecular mass of 65 kDa. Moreimportant, the mature plant protein is catalytically active. In the invitro enzyme assay described below, the purified recombinant spinach RSexhibited a turnover number of approximately 0.08/sec at 25° C. (basedon protomer). By way of comparison, the reported turnover numbers for S.cerevisiae (Santos et al., J. Biol. Chem. 270:437-444 (1995)) and B.subtilis (Bacher et al., J. Biol. Chem. 255:632-637 (1980)) RS, at 37°C., are 0.13/sec and 0.33/sec, respectively. Assuming that the enzymereaction is characterized by a Q10 (temperature coefficient) of at least2, these observations suggest that the purified recombinant spinach RSis probably fully active.

EXAMPLE 7 Purification and Physical Properties of Recombinant MatureSpinach LS

An aliquot (0.5 mL) of E. coli cell-free extract containing therecombinant spinach LS was rapidly thawed to room temperature, diluted1:1 with deionized water, and filtered through a 0.2 μm Acrodisc filter(Gelman Sciences, Cat. No. 4192). The entire sample was thenfractionated on a Mono Q HR 5/5 column, using the same buffers andconditions that were described above for recombinant spinach RS. Thematerial eluting between 0.367-0.433 M NaCl was pooled, concentrated to450 μL, and subjected to gel filtration chromatography exactly asdescribed above for the recombinant spinach RS. Highly purified spinachLS emerged from the sizing column as a sharp peak eluting between10.15-10.85 min, and this material was supplemented with glycerol (5%),concentrated to 12.1 mg of protein/mL, and stored at −80° C. forsubsequent use. The final yield of purified protein was 4.3 mg (nearly22% of the total protein present in the cell-free extract) and thepreparation was essentially homogeneous as judged from Coomassie-stainedgels.

The nucleotide sequence of the mature recombinant spinach LS predicts apolypeptide of 16534.71 daltons, a value that is virtually identical tothat which was obtained with the purified protein using electrosprayionization mass spectrometry (16536.3 daltons). Assuming that plant LSforms a hollow, spherical particle, comprised of 60 identical subunits,like the E. coli (Mörtl et al., J. Biol. Chem. 271:33201-33207 (1996))and B. subtilis (Bacher et al., J. Biol Chem. 255:632-637 (1980))homologs, its native molecular mass should be about 992 kDa. Indeed,during analytical gel filtration, the purified recombinant spinach LSexhibited an apparent molecular mass of about 823 kDa. It would thusappear that the quaternary structure of this riboflavin biosyntheticenzyme has been highly conserved in the evolution from bacteria tohigher plants. Edman degradation confirmed that the first two N-terminalamino acid residues of the recombinant protein had been correctlyaltered through PCR from Val-Arg to Met-Asn as previously described.Despite these substitutions and removal of the chloroplast targetingsequence, the purified recombinant spinach LS still retained catalyticactivity. At 25° C., using the in vitro enzyme assay described below,its turnover number was 0.013/sec (based on protomer). This value is inreasonable agreement with the turnover numbers reported for the purifiedE. coli, yeast, and B. subtilis enzymes (0.06/sec), which were allmeasured at 37° C. (Kis et al., Biochemistry 34:2883-2892 (1995); Mörtlet al., J. Biol. Chem. 271 :33201-33207 (1996)). Thus, it is very likelythat the recombinant spinach LS is also fully active.

EXAMPLE 8 Preparation of Substrates For RS and LS

6,7-Dimethyl-8-(1′-D-ribityl)lumazine (DMRL) was synthesized aspreviously described (Plaut, G. W. E. and Harvey, R. A., Methods inEnzvmology (McCormick, D. B and Wright L. D., eds.) vol. 18, part B, pp.515-538, Academic Press, NY (1971)) and purified by HPLC on a C-18column developed with a water to methanol gradient. The purifiedmaterial was taken to dryness in a rotovap and stored at −20° C. forsubsequent use.

4-Ribitylamino-5-amino-2,6-dihydroxypyrimidine (RAADP) was prepared from4-ribitylamino-5-nitroso-2,6-dihydroxypyrimidine by catalytichydrogenation (Plaut, G. W. E. and Harvey, R. A., Methods in Enzymology(McCormick, D. B and Wright L. D., eds.) vol. 18, part B, pp. 515-538,Academic Press, NY (1971)). The 40-mL reaction mixture contained 0.4mmol of the latter compound, dissolved in 10 mM acetic acid, and 20 mgof 10% palladium on carbon. Following an overnight incubation period at25° C. (with gentle shaking, under 50 psi H₂) the catalyst was removedby filtration and the filtrate containing RAADP was stored in aliquotsat −80° C.

3,4-Dihydroxybutanone 4-phosphate (DHBP) was prepared enzymatically fromD-ribose 5-phosphate. The reaction mixture contained 50 mM Tris-HCl (pH7.5), 20 mM MgCl₂, 20 mM D-ribose 5-phosphate, 10 units/mLphosphoribo-isomerase (Sigma, Cat. No. P7434) and 0.3 units/mL E. coliDHBP synthase. After 2 h at 25° C., the reaction reached completion andaliquots of the solution containing DHBP were stored at −80° C.

EXAMPLE 9 Riboflavin Synthase Assays

Riboflavin synthase assays were run using 1-mL reaction mixturescontaining 0.1 mM DMRL in 50 mM Tris-Cl (pH 7.5) at 25° C. Reactionswere initiated by the addition of purified recombinant spinach RS andinitial rates of the reactions were measured continuously at 470 nm. Amolar extinction coefficient (ε) of 9500 at 470 nm was used to calculatethe formation of riboflavin (Plaut, G. W. E. and Harvey, R. A., Methodsin Enzymology (McCormick, D. B. and Wright L. D., eds.) vol. 18, part B,pp. 515-538, Academic Press, NY (1971)).

Inhibitor screens were carried out in 96-well plates. Potentialinhibitory compounds were dissolved in dimethyl sulfoxide (DMSO) (10mg/mL) and then serially diluted with water to concentrations of 0.1mg/mL in 1% aqueous DMSO. Reaction mixtures (0.21 mL total) contained0.115 mL DMRL (0.035 mM) in 100 mM Tris-Cl (pH 7.5) and 0.085 mLpotential inhibitory compound (0.1 mg/mL) at 25° C. Before initiatingreactions with enzyme, the absorbance at 470 nm was recorded. Reactionswere initiated with 0.01 mL of purified recombinant spinach RS (0.28mg/mL) and after a 3 min incubation at 25° C. the plates were read at470 nm. The first absorbance reading is subtracted from the second toafford rates of riboflavin formation. Column 1 of the 96-well platescontained no compounds and the reactions in these wells served asuninhibited controls. Compounds that reduced the rate of riboflavinformation (in comparison to the uninhibited control reactions) werefollowed up with 1-mL confirmation assays where IC50's were determined.

EXAMPLE 10 Lumazine Synthase Assays

Lumazine synthase assays were run using 1-mL reaction mixtures whichcontain 0.05 mM RAADP and 0.05 mM DHBP in 50 mM Tris-HCl (pH 7.5) at 25°C. Reactions were then initiated by the addition of purified recombinantspinach LS and initial rates of the reactions were monitoredcontinuously at an absorbance of 408 nm. A molar extinction coefficient(ε) of 10,000 at 408 nm was used to calculate the rate of formation ofDMRL (Plaut, G. W. E. and Harvey, R. A., Methods in Enzymology(McCormick, D. B and Wright L. D., eds.) vol. 18, part B, pp. 515-538,Academic Press, NY (1971)).

Inhibitor screens are carried out in 96-well plates. Potentialinhibitory compounds are dissolved in 10% aqueous DMSO to aconcentration of 1 mg/mL. Reaction mixtures (0.21 mL total) are preparedby adding 0.005 mL potential inhibitory compound (1.0 mg/mL) to 0.195 mLof a solution containing 0.05 mM DHBP, 0.05 mM RAADP in 50 mM Tris-Cl(pH 7.5) at 25° C. Reactions are then initiated with 0.01 mL of purifiedrecombinant spinach LS (1.6 mg/mL) and progress of the reactions ismonitored continuously at 408 nm for 5 min. Column 1 of the 96-wellplates contains no test compounds and the rates of DMRL formation inthese wells serve as uninhibited controls. Compounds that reduce therates of DMPL formation (in comparison to the uninhibited controls) arefollowed up with 1-mL confirmation assays where IC50's are determined.

EXAMPLE 11 Other Plant and Fungal RS and LS Genes

Using the methodologies described in Example 4, several other plant andfungal LS and RS cDNAs have been cloned through functionalcomplementation of the E. coli riboflavin auxotrophs. These include thenuclear-encoded precursor proteins for arabidopsis RS, tobacco LS, andarabidopsis LS and full-length mature RS and LS proteins from the riceblast fungus Magnaporthe grisea. The nucleotide sequences of the ORFsfor these proteins are respectively documented in SEQ ID NO:35, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:11 and SEQ ID NO:37, and theircorresponding amino acid sequences are given in SEQ ID NO:36, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:12, and SEQ ID NO:38, respectively. TheLamba cDNA expression libraries from which the arabidopsis LS and RSgenes and tobacco LS gene were cloned are commercially available fromStratagene (Cat. Nos. 937010 and 936002, respectively). The cDNAexpression library from which the Magnaporthe grisea RS gene wasobtained was contained in the Lambda ZipLox vector (GibcoBRL) betweenthe NotI and SalI cleavage sites of the polylinker region, and wasprepared from isolated mRNA using conventional methodologies (Maniatis).The cDNA expression library from which the Magnaporthe grisea LS genewas obtained was cloned between the EcoRI and XhoI sites of the plasmidvector pBluescript II SK (+), available from Stratagene, and was alsoprepared from isolated mRNA using standard procedures (Maniatis). Asdescribed above for cloning the spinach LS and RS precursor genes (as inExample 4), the various Lamba cDNA expression libraries were firstsubjected to mass excision to yield plasmid cDNA libraries, which werethen introduced into the E. coli LS and RS auxotrophs (W3110derivatives) via electroporation. Following this procedure,transformants were selected for growth in the absence of addedriboflavin (on plates containing B agar), and the cDNA inserts of therescuing plasmids were isolated and sequenced completely usingcustom-designed primers.

As shown in FIG. 3, the cloned spinach and arabidopsis RS are verysimilar. Both proteins are synthesized as larger molecular weightprecursors with a chloroplast targeting sequence at their N-terminus(boxed residues in FIG. 3). Although the transit peptides of the twoplant species are of comparable length, they are highly divergent andbear little resemblance to each other at the primary amino sequencelevel. In contrast, the mature spinach and arabidopsis RS are wellconserved, with nearly 70% (as determined by the GCG “Gap” program) oftheir residues being identical. These observations are not surprisingsince most nuclear-encoded chloroplast proteins, including the precursorfor the small subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase(Mazur et al., Nucl. Acids Res. 13:2373-2386 (1985)), exhibit muchgreater species-to-species variation in their chloroplast targetingsequence than in the mature portion of the molecule. The two plant RShomologs also possess a number of polypeptide motifs that are notpresent in the bacterial and fungal RS homologs that have currently beensequenced. It is possible that some of these highly conserved regionsthat are unique to plants might specifically influence the catalyticand/or regulatory properties of the higher plant RS, thereby providingvaluable insight in the design of enzyme inhibitors that could be usefulas herbicides.

A comparison of the spinach, tobacco, and arabidopsis LS precursorproteins, also cloned by functional complementation, provides a similarpicture (FIG. 4). Again, the chloroplast transit peptides of threeprecursors are poorly conserved (boxed residues), while the matureproteins exhibit 72-76% identity at the amino acid sequence level, asdetermined by the GCG “Gap” program. Additionally, all three plantproteins possess a unique stretch of amino acid residues at theirC-terminus (e.g., ASLFEHHLK [SEQ ID NO:39]), a region of the polypeptidethat is highly divergent in microbial LS homologs. That these nineresidues are identical for three different plant species, suggests thatthey might be of unique importance to the functionality of the higherplant proteins. Based on this observation, it is anticipated thatfurther structural and mechanistic studies with the purified plant LSproteins described herein will greatly assist the rational design ofenzyme inhibitors that are specifically herbicidal.

Until the present invention, the only fungal LS and RS homologs thathave been sequenced are those of S. cervasiae (Garcia-Ramirez et al., J.Biol. Chem. 270:23801-23807 (1995)); Santos et al., J. Biol. Chem.270:437-444 (1995)). These proteins bear little resemblance to theMagnaporthe grisea LS and RS proteins that were cloned in the presentwork by functional complementation. The two fungal RS homologs are only47% identical at the primary amino acid sequence level. While a similardegree of conservation is observed between the Magnaporthe grisea and S.cerevesiae LS proteins (51% identity), the former is significantlylonger than other known microbial homologs. Moreover, the additionallength of this protein is not due to the presence of a cleavableN-terminal targeting sequence as described above for the higher plant LSand RS precursors. Instead, it possesses a unique polypeptide segmentthat appears to have been inserted right about in the middle of theprotein; based on sequence alignments with other LS homologs, theadditional residues (consisting largely of Ser and Thr) correspond toamino acids 74-104 of the Magnaporthe grisea LS (SEQ ID NO:38). Thesignificance of this observation is not yet understood, but it couldhave practical application in the development of novel fungicides foruse in the treatment of rice blast.

39 1 471 DNA Spinacia sp. 1 gttagggagc ttgaaggtta tgtcactaaa gcccagagtttcagatttgc cattgttgtg 60 gctaggttca acgaatttgt gacaagacga ctaatggaaggagctcttga cacttttaag 120 aaatactctg tcaatgaaga tattgatgtt gtttgggttcctggtgctta tgagctaggt 180 gttactgcac aagcacttgg gaaatcagga aaatatcatgctattgtttg tcttggagct 240 gtggtaaaag gtgatacttc acactatgat gctgtcgttaattctgcttc ctctggagta 300 ctgtcagctg gattaaattc aggagtacct tgtgtctttggtgtccttac ctgtgataac 360 atggatcagg ccataaatcg agctggcggg aaagcgggtaataaaggagc cgagtcagcg 420 ctaacagcta ttgaaatggc ttcgcttttc gaacatcatttgaaggccta a 471 2 156 PRT Spinacia sp. 2 Val Arg Glu Leu Glu Gly TyrVal Thr Lys Ala Gln Ser Phe Arg Phe 1 5 10 15 Ala Ile Val Val Ala ArgPhe Asn Glu Phe Val Thr Arg Arg Leu Met 20 25 30 Glu Gly Ala Leu Asp ThrPhe Lys Lys Tyr Ser Val Asn Glu Asp Ile 35 40 45 Asp Val Val Trp Val ProGly Ala Tyr Glu Leu Gly Val Thr Ala Gln 50 55 60 Ala Leu Gly Lys Ser GlyLys Tyr His Ala Ile Val Cys Leu Gly Ala 65 70 75 80 Val Val Lys Gly AspThr Ser His Tyr Asp Ala Val Val Asn Ser Ala 85 90 95 Ser Ser Gly Val LeuSer Ala Gly Leu Asn Ser Gly Val Pro Cys Val 100 105 110 Phe Gly Val LeuThr Cys Asp Asn Met Asp Gln Ala Ile Asn Arg Ala 115 120 125 Gly Gly LysAla Gly Asn Lys Gly Ala Glu Ser Ala Leu Thr Ala Ile 130 135 140 Glu MetAla Ser Leu Phe Glu His His Leu Lys Ala 145 150 155 3 474 DNA TOBACCO 3gttcgtcagt tgactggttc tgttacctct gccaaaggcc atcgctttgc tgttgtggtt 60gcacgtttca atgatcttat caccaagaag cttttggagg gagctttgga cactttcaaa 120aattactctg ttagagagga agatattgat gtcgtgtggg ttcctggctg ttttgaaatc 180ggtgtggttg cgcaacagct tggaaagtcg cagaaatatc aagcaatact ctgtattggg 240gctgtgatta gaggtgatac gtctcactat gatgccgtcg ttaatgctgc cacatccgga 300gtactttcag caggtctaaa ttctggtact ccttgcatat ttggtgtttt gacatgtgat 360accttggagc aggctttcaa tcgtgtcggt gggaaggctg ggaataaagg tgccgaaaca 420gcgttgacag ctattgagat ggcgtctttg tttgaacacc acttaaaggc ttaa 474 4 157PRT TOBACCO 4 Val Arg Gln Leu Thr Gly Ser Val Thr Ser Ala Lys Gly HisArg Phe 1 5 10 15 Ala Val Val Val Ala Arg Phe Asn Asp Leu Ile Thr LysLys Leu Leu 20 25 30 Glu Gly Ala Leu Asp Thr Phe Lys Asn Tyr Ser Val ArgGlu Glu Asp 35 40 45 Ile Asp Val Val Trp Val Pro Gly Cys Phe Glu Ile GlyVal Val Ala 50 55 60 Gln Gln Leu Gly Lys Ser Gln Lys Tyr Gln Ala Ile LeuCys Ile Gly 65 70 75 80 Ala Val Ile Arg Gly Asp Thr Ser His Tyr Asp AlaVal Val Asn Ala 85 90 95 Ala Thr Ser Gly Val Leu Ser Ala Gly Leu Asn SerGly Thr Pro Cys 100 105 110 Ile Phe Gly Val Leu Thr Cys Asp Thr Leu GluGln Ala Phe Asn Arg 115 120 125 Val Gly Gly Lys Ala Gly Asn Lys Gly AlaGlu Thr Ala Leu Thr Ala 130 135 140 Ile Glu Met Ala Ser Leu Phe Glu HisHis Leu Lys Ala 145 150 155 5 471 DNA arabidopsis 5 gttcgccatgttacggggtc tcttatcaga ggcgaaggtc ttagattcgc catcgtggta 60 gctcgtttcaatgaggttgt gactaagttg cttttggaag gagcgattga gactttcaag 120 aagtattcagtcagagaaga agacattgaa gttatttggg ttcctggcag ctttgaaatt 180 ggtgttgttgcacaaaatct tgggaaatcg ggaaaatttc atgctgtttt atgtatcggc 240 gctgtgataagaggagatac cacacattat gatgctgttg ccaactctgc tgcgtctgga 300 gtactttctgctagcataaa ttcaggcgtt ccatgcatat ttggtgtact gacttgcgag 360 gacatggatcaggctctgaa tcgatctggt ggcaaagccg gcaataaggg agctgaaact 420 gctttgacggcgctcgaaat ggcgtcgttg tttgagcacc acctgaaata g 471 6 156 PRT arabidopsis6 Val Arg His Val Thr Gly Ser Leu Ile Arg Gly Glu Gly Leu Arg Phe 1 5 1015 Ala Ile Val Val Ala Arg Phe Asn Glu Val Val Thr Lys Leu Leu Leu 20 2530 Glu Gly Ala Ile Glu Thr Phe Lys Lys Tyr Ser Val Arg Glu Glu Asp 35 4045 Ile Glu Val Ile Trp Val Pro Gly Ser Phe Glu Ile Gly Val Val Ala 50 5560 Gln Asn Leu Gly Lys Ser Gly Lys Phe His Ala Val Leu Cys Ile Gly 65 7075 80 Ala Val Ile Arg Gly Asp Thr Thr His Tyr Asp Ala Val Ala Asn Ser 8590 95 Ala Ala Ser Gly Val Leu Ser Ala Ser Ile Asn Ser Gly Val Pro Cys100 105 110 Ile Phe Gly Val Leu Thr Cys Glu Asp Met Asp Gln Ala Leu AsnArg 115 120 125 Ser Gly Gly Lys Ala Gly Asn Lys Gly Ala Glu Thr Ala LeuThr Ala 130 135 140 Leu Glu Met Ala Ser Leu Phe Glu His His Leu Lys 145150 155 7 633 DNA Spinach 7 atgttcactg gcattgttga agagattggc cgagttaagcaaatgggtta tggcgaagac 60 ggtggatttc agcttaaagt tgtaggagac attgtcctaaaagatgtcaa tcttggtgac 120 agtatcgcag ttaatggtac atgtctaact gtgacggaatttgacactaa agcgtccgaa 180 tttactcttg ggatagcgcc tgagacgctt aggaagacggcattgatgga tctcgaacca 240 gggtcagttg ttaatttaga aagagccctt ttgccttctacacggatggg tggtcacttt 300 gtccagggac atgttgatgg gacaggagaa attgtatcactagttgaaga aggtgattct 360 ttgtgggtca agataaaaac aagcccagaa atactgagatacattgtacc aaaagggttt 420 attgcaattg atggcacaag tttaacagtg gtggatgtgtttgaccaaga attatgcttt 480 aatattatgt tagttgctta cactcaacaa aatgtggtcattccactcaa aaaagttggc 540 caaaaggtta atttagaggt tgatattcta ggaaaatatgtggaaaggct cctaagtagt 600 agtggggttt tggatcctac caaattcaca tag 633 8 210PRT Spinach 8 Met Phe Thr Gly Ile Val Glu Glu Ile Gly Arg Val Lys GlnMet Gly 1 5 10 15 Tyr Gly Glu Asp Gly Gly Phe Gln Leu Lys Val Val GlyAsp Ile Val 20 25 30 Leu Lys Asp Val Asn Leu Gly Asp Ser Ile Ala Val AsnGly Thr Cys 35 40 45 Leu Thr Val Thr Glu Phe Asp Thr Lys Ala Ser Glu PheThr Leu Gly 50 55 60 Ile Ala Pro Glu Thr Leu Arg Lys Thr Ala Leu Met AspLeu Glu Pro 65 70 75 80 Gly Ser Val Val Asn Leu Glu Arg Ala Leu Leu ProSer Thr Arg Met 85 90 95 Gly Gly His Phe Val Gln Gly His Val Asp Gly ThrGly Glu Ile Val 100 105 110 Ser Leu Val Glu Glu Gly Asp Ser Leu Trp ValLys Ile Lys Thr Ser 115 120 125 Pro Glu Ile Leu Arg Tyr Ile Val Pro LysGly Phe Ile Ala Ile Asp 130 135 140 Gly Thr Ser Leu Thr Val Val Asp ValPhe Asp Gln Glu Leu Cys Phe 145 150 155 160 Asn Ile Met Leu Val Ala TyrThr Gln Gln Asn Val Val Ile Pro Leu 165 170 175 Lys Lys Val Gly Gln LysVal Asn Leu Glu Val Asp Ile Leu Gly Lys 180 185 190 Tyr Val Glu Arg LeuLeu Ser Ser Ser Gly Val Leu Asp Pro Thr Lys 195 200 205 Phe Thr 210 9627 DNA arabidopsis 9 gtgtttactg gaatcgtgga ggaaatgggt gaagtcaaggacttgggaat ggccgatcac 60 ggaggattcg acctcaaaat cggagcgaga gtggtgttagaggacgtgaa gctcggtgac 120 agtatcgccg tgaacggtac ttgtttaacg gtgacggagtttaacgcaga ggagttcaca 180 gtagggttag caccggagac gctgagaaaa acatcgttggaggagttaaa gaaaggatct 240 ccggtgaatc tggagcgtgc gttgcagcca gtgagcaggatgggtggaca cgtggttcag 300 ggacacgtgg atgggacggg agtgattgaa tcaatggaggtagagggtga ttctttgtgg 360 gtgaaggtga aagctgacaa gggtttgttg aaatacattgtgcctaaagg atttgtggct 420 gttgatggga ctagcttgac ggttgttgat gtgtttgatgaagagagctg tttcaatttc 480 atgatgattg cttatacgca acagaatgta gtgattccgactaagaagat tgggcagaaa 540 gtgaatcttg aggttgatat catggggaag tatgttgagaggcttctcac cagtggtggc 600 ttctccaaag gaaaagaaaa tatttga 627 10 208 PRTarabidopsis 10 Val Phe Thr Gly Ile Val Glu Glu Met Gly Glu Val Lys AspLeu Gly 1 5 10 15 Met Ala Asp His Gly Gly Phe Asp Leu Lys Ile Gly AlaArg Val Val 20 25 30 Leu Glu Asp Val Lys Leu Gly Asp Ser Ile Ala Val AsnGly Thr Cys 35 40 45 Leu Thr Val Thr Glu Phe Asn Ala Glu Glu Phe Thr ValGly Leu Ala 50 55 60 Pro Glu Thr Leu Arg Lys Thr Ser Leu Glu Glu Leu LysLys Gly Ser 65 70 75 80 Pro Val Asn Leu Glu Arg Ala Leu Gln Pro Val SerArg Met Gly Gly 85 90 95 His Val Val Gln Gly His Val Asp Gly Thr Gly ValIle Glu Ser Met 100 105 110 Glu Val Glu Gly Asp Ser Leu Trp Val Lys ValLys Ala Asp Lys Gly 115 120 125 Leu Leu Lys Tyr Ile Val Pro Lys Gly PheVal Ala Val Asp Gly Thr 130 135 140 Ser Leu Thr Val Val Asp Val Phe AspGlu Glu Ser Cys Phe Asn Phe 145 150 155 160 Met Met Ile Ala Tyr Thr GlnGln Asn Val Val Ile Pro Thr Lys Lys 165 170 175 Ile Gly Gln Lys Val AsnLeu Glu Val Asp Ile Met Gly Lys Tyr Val 180 185 190 Glu Arg Leu Leu ThrSer Gly Gly Phe Ser Lys Gly Lys Glu Asn Ile 195 200 205 11 645 DNAMagnaporthe grisea 11 atgttcactg gtatagtcga ggagatcgga gtcgtggccgagctcaaccc gcacgatgcc 60 actggaggga cgtcattgac catctcgctc ccgacgggcagcagcctgct ctcggattgc 120 cacgacggtg atagcatcgc cgtcaacggt gtgtgcctgaccgtcacatc cttcacgccg 180 acgcagttca cagtcggtgt tgccccggag acgctgcgcgtcacggacct gggcgacctg 240 gtcaaggact cgcgcgtgaa cctggagcga gccgtgcgggccgacactcg catgggcggt 300 cactttgtac agggccacgt cgacacgacc gccaccatagccgacaagca ggcagatggt 360 aacgccgtca cgatgcggtt caagccacgg gagggtagcgatgtgttgaa gtacatcgtg 420 cgaaagggtt atgtcgcatt ggacggaacc agcttgacggttactaaggt cgacgacgct 480 gccgggtggt gggaggtcat gctcatcgtt tacacgcaggaacgtgtggt cctggcgcag 540 aagaacgttg gtgatactgt caatgtcgag gttgacgtcttggccaagta tgctgagaag 600 agtatggctg gatacttgag ctctctcaac aagagtgacgcataa 645 12 214 PRT Magnaporthe grisea 12 Met Phe Thr Gly Ile Val GluGlu Ile Gly Val Val Ala Glu Leu Asn 1 5 10 15 Pro His Asp Ala Thr GlyGly Thr Ser Leu Thr Ile Ser Leu Pro Thr 20 25 30 Gly Ser Ser Leu Leu SerAsp Cys His Asp Gly Asp Ser Ile Ala Val 35 40 45 Asn Gly Val Cys Leu ThrVal Thr Ser Phe Thr Pro Thr Gln Phe Thr 50 55 60 Val Gly Val Ala Pro GluThr Leu Arg Val Thr Asp Leu Gly Asp Leu 65 70 75 80 Val Lys Asp Ser ArgVal Asn Leu Glu Arg Ala Val Arg Ala Asp Thr 85 90 95 Arg Met Gly Gly HisPhe Val Gln Gly His Val Asp Thr Thr Ala Thr 100 105 110 Ile Ala Asp LysGln Ala Asp Gly Asn Ala Val Thr Met Arg Phe Lys 115 120 125 Pro Arg GluGly Ser Asp Val Leu Lys Tyr Ile Val Arg Lys Gly Tyr 130 135 140 Val AlaLeu Asp Gly Thr Ser Leu Thr Val Thr Lys Val Asp Asp Ala 145 150 155 160Ala Gly Trp Trp Glu Val Met Leu Ile Val Tyr Thr Gln Glu Arg Val 165 170175 Val Leu Ala Gln Lys Asn Val Gly Asp Thr Val Asn Val Glu Val Asp 180185 190 Val Leu Ala Lys Tyr Ala Glu Lys Ser Met Ala Gly Tyr Leu Ser Ser195 200 205 Leu Asn Lys Ser Asp Ala 210 13 38 DNA Artificial SequenceDescription of Artificial SequencePRIMER 13 cgaaggaaga ccatggccattattgaagct aacgttgc 38 14 34 DNA Artificial Sequence Description ofArtificial SequencePRIMER 14 atcttactgt cgacttcagg ccttgatggc tttc 34 1535 DNA Artificial Sequence Description of Artificial SequencePRIMER 15actcatttac catggctacg gggattgtac agggc 35 16 35 DNA Artificial SequenceDescription of Artificial SequencePRIMER 16 atcttactgt cgacttcaggcttctgtgcc tggtt 35 17 37 DNA Artificial Sequence Description ofArtificial SequencePRIMER 17 aactagatca gcggccgcag ccacgttgtg tctcaaa 3718 36 DNA Artificial Sequence Description of Artificial SequencePRIMER18 gacaaacata gcggccgctg aggtctgcct cgtgaa 36 19 36 DNA ArtificialSequence Description of Artificial SequencePRIMER 19 aactagatcagcggccgcgg tacggttatt cgtggt 36 20 33 DNA Artificial SequenceDescription of Artificial SequencePRIMER 20 gacaaacata gcggccgcgtcgtatttacc ggt 33 21 35 DNA Artificial Sequence Description ofArtificial SequencePRIMER 21 aactagatca gcggccgcac cactgctgaa gtggc 3522 37 DNA Artificial Sequence Description of Artificial SequencePRIMER22 gacaaacata gcggccgcga cctgacatta agtgtcc 37 23 34 DNA ArtificialSequence Description of Artificial SequencePRIMER 23 ctactcatttcatatgttca ctggcattgt tgaa 34 24 37 DNA Artificial Sequence Descriptionof Artificial SequencePRIMER 24 catcttactg gatccactat gtgaatttgg taggatc37 25 39 DNA Artificial Sequence Description of ArtificialSequencePRIMER 25 ctactcattt catatgaacg agcttgaagg ttatgtcac 39 26 38DNA Artificial Sequence Description of Artificial SequencePRIMER 26catcttactg gatccatcag gccttcaaat gatgttcg 38 27 669 DNA spinach 27atggcttcat ttgcagcttc tcaaacttgt ttcctgacaa caaaccccac ttgtttaaaa 60cccaattccc ctcaaaaatc ttccacattt cttccatttt ctgcccctct ttcttcctcg 120tcatctttcc ctggttgtgg gttggttcat gttgcatcaa acaagaaaaa tcgtgcttcg 180tttgtagtga ccaatgctgt tagggagctt gaaggttatg tcactaaagc ccagagtttc 240agatttgcca ttgttgtggc taggttcaac gaatttgtga caagacgact aatggaagga 300gctcttgaca cttttaagaa atactctgtc aatgaagata ttgatgttgt ttgggttcct 360ggtgcttatg agctaggtgt tactgcacaa gcacttggga aatcaggaaa atatcatgct 420attgtttgtc ttggagctgt ggtaaaaggt gatacttcac actatgatgc tgtcgttaat 480tctgcttcct ctggagtact gtcagctgga ttaaattcag gagtaccttg tgtctttggt 540gtccttacct gtgataacat ggatcaggcc ataaatcgag ctggcgggaa agcgggtaat 600aaaggagccg agtcagcgct aacagctatt gaaatggctt cgcttttcga acatcatttg 660aaggcctaa 669 28 222 PRT spinach 28 Met Ala Ser Phe Ala Ala Ser Gln ThrCys Phe Leu Thr Thr Asn Pro 1 5 10 15 Thr Cys Leu Lys Pro Asn Ser ProGln Lys Ser Ser Thr Phe Leu Pro 20 25 30 Phe Ser Ala Pro Leu Ser Ser SerSer Ser Phe Pro Gly Cys Gly Leu 35 40 45 Val His Val Ala Ser Asn Lys LysAsn Arg Ala Ser Phe Val Val Thr 50 55 60 Asn Ala Val Arg Glu Leu Glu GlyTyr Val Thr Lys Ala Gln Ser Phe 65 70 75 80 Arg Phe Ala Ile Val Val AlaArg Phe Asn Glu Phe Val Thr Arg Arg 85 90 95 Leu Met Glu Gly Ala Leu AspThr Phe Lys Lys Tyr Ser Val Asn Glu 100 105 110 Asp Ile Asp Val Val TrpVal Pro Gly Ala Tyr Glu Leu Gly Val Thr 115 120 125 Ala Gln Ala Leu GlyLys Ser Gly Lys Tyr His Ala Ile Val Cys Leu 130 135 140 Gly Ala Val ValLys Gly Asp Thr Ser His Tyr Asp Ala Val Val Asn 145 150 155 160 Ser AlaSer Ser Gly Val Leu Ser Ala Gly Leu Asn Ser Gly Val Pro 165 170 175 CysVal Phe Gly Val Leu Thr Cys Asp Asn Met Asp Gln Ala Ile Asn 180 185 190Arg Ala Gly Gly Lys Ala Gly Asn Lys Gly Ala Glu Ser Ala Leu Thr 195 200205 Ala Ile Glu Met Ala Ser Leu Phe Glu His His Leu Lys Ala 210 215 22029 678 DNA tobacco 29 ttcgctttcg gacagtgcaa tcttctacct cgtacaacaactgtaaatcc cacacaactg 60 cactctcctc tttactcttt gtctttgcct ttccacagacaaagcataac ctcttcacct 120 gcactatcat tcacccaatc tcaaggttta gggtctgcaattgagagaca ttgcgaccgg 180 tcggatctgt ttcaaacatg tgctgttcgt cagttgactggttctgttac ctctgccaaa 240 ggccatcgct ttgctgttgt ggttgcacgt ttcaatgatcttatcaccaa gaagcttttg 300 gagggagctt tggacacttt caaaaattac tctgttagagaggaagatat tgatgtcgtg 360 tgggttcctg gctgttttga aatcggtgtg gttgcgcaacagcttggaaa gtcgcagaaa 420 tatcaagcaa tactctgtat tggggctgtg attagaggtgatacgtctca ctatgatgcc 480 gtcgttaatg ctgccacatc cggagtactt tcagcaggtctaaattctgg tactccttgc 540 atatttggtg ttttgacatg tgataccttg gagcaggctttcaatcgtgt cggtgggaag 600 gctgggaata aaggtgccga aacagcgttg acagctattgagatggcgtc tttgtttgaa 660 caccacttaa aggcttaa 678 30 225 PRT tobacco 30Phe Ala Phe Gly Gln Cys Asn Leu Leu Pro Arg Thr Thr Thr Val Asn 1 5 1015 Pro Thr Gln Leu His Ser Pro Leu Tyr Ser Leu Ser Leu Pro Phe His 20 2530 Arg Gln Ser Ile Thr Ser Ser Pro Ala Leu Ser Phe Thr Gln Ser Gln 35 4045 Gly Leu Gly Ser Ala Ile Glu Arg His Cys Asp Arg Ser Asp Leu Phe 50 5560 Gln Thr Cys Ala Val Arg Gln Leu Thr Gly Ser Val Thr Ser Ala Lys 65 7075 80 Gly His Arg Phe Ala Val Val Val Ala Arg Phe Asn Asp Leu Ile Thr 8590 95 Lys Lys Leu Leu Glu Gly Ala Leu Asp Thr Phe Lys Asn Tyr Ser Val100 105 110 Arg Glu Glu Asp Ile Asp Val Val Trp Val Pro Gly Cys Phe GluIle 115 120 125 Gly Val Val Ala Gln Gln Leu Gly Lys Ser Gln Lys Tyr GlnAla Ile 130 135 140 Leu Cys Ile Gly Ala Val Ile Arg Gly Asp Thr Ser HisTyr Asp Ala 145 150 155 160 Val Val Asn Ala Ala Thr Ser Gly Val Leu SerAla Gly Leu Asn Ser 165 170 175 Gly Thr Pro Cys Ile Phe Gly Val Leu ThrCys Asp Thr Leu Glu Gln 180 185 190 Ala Phe Asn Arg Val Gly Gly Lys AlaGly Asn Lys Gly Ala Glu Thr 195 200 205 Ala Leu Thr Ala Ile Glu Met AlaSer Leu Phe Glu His His Leu Lys 210 215 220 Ala 225 31 684 DNAarabidopsis 31 atgaagtcat tagcttcgcc gccgtgtctc cgcctgatac cgacggcacaccgtcagctc 60 aattcgcgtc aatcttcctc cgcctgttat atacacggtg gctcttctgtgaacaaatcc 120 aataatctct cattctcctc atccacatcc ggatttgcgt caccactagctgtagagaag 180 gaattacgct cttcattcgt acagacggct gctgttcgcc atgttacggggtctcttatc 240 agaggcgaag gtcttagatt cgccatcgtg gtagctcgtt tcaatgaggttgtgactaag 300 ttgcttttgg aaggagcgat tgagactttc aagaagtatt cagtcagagaagaagacatt 360 gaagttattt gggttcctgg cagctttgaa attggtgttg ttgcacaaaatcttgggaaa 420 tcgggaaaat ttcatgctgt tttatgtatc ggcgctgtga taagaggagataccacacat 480 tatgatgctg ttgccaactc tgctgcgtct ggagtacttt ctgctagcataaattcaggc 540 gttccatgca tatttggtgt actgacttgc gaggacatgg atcaggctctgaatcgatct 600 ggtggcaaag ccggcaataa gggagctgaa actgctttga cggcgctcgaaatggcgtcg 660 ttgtttgagc accacctgaa atag 684 32 227 PRT arabidopsis 32Met Lys Ser Leu Ala Ser Pro Pro Cys Leu Arg Leu Ile Pro Thr Ala 1 5 1015 His Arg Gln Leu Asn Ser Arg Gln Ser Ser Ser Ala Cys Tyr Ile His 20 2530 Gly Gly Ser Ser Val Asn Lys Ser Asn Asn Leu Ser Phe Ser Ser Ser 35 4045 Thr Ser Gly Phe Ala Ser Pro Leu Ala Val Glu Lys Glu Leu Arg Ser 50 5560 Ser Phe Val Gln Thr Ala Ala Val Arg His Val Thr Gly Ser Leu Ile 65 7075 80 Arg Gly Glu Gly Leu Arg Phe Ala Ile Val Val Ala Arg Phe Asn Glu 8590 95 Val Val Thr Lys Leu Leu Leu Glu Gly Ala Ile Glu Thr Phe Lys Lys100 105 110 Tyr Ser Val Arg Glu Glu Asp Ile Glu Val Ile Trp Val Pro GlySer 115 120 125 Phe Glu Ile Gly Val Val Ala Gln Asn Leu Gly Lys Ser GlyLys Phe 130 135 140 His Ala Val Leu Cys Ile Gly Ala Val Ile Arg Gly AspThr Thr His 145 150 155 160 Tyr Asp Ala Val Ala Asn Ser Ala Ala Ser GlyVal Leu Ser Ala Ser 165 170 175 Ile Asn Ser Gly Val Pro Cys Ile Phe GlyVal Leu Thr Cys Glu Asp 180 185 190 Met Asp Gln Ala Leu Asn Arg Ser GlyGly Lys Ala Gly Asn Lys Gly 195 200 205 Ala Glu Thr Ala Leu Thr Ala LeuGlu Met Ala Ser Leu Phe Glu His 210 215 220 His Leu Lys 225 33 840 DNASpinach 33 atggcacttt caacttcact ctctttagta tctcccaaac tctctcaacaaaatctcaca 60 ttttgcacct tcaacaacca accctcctct ttaaatgggc atatcaaattcaatccaaac 120 ctcagaaact cagtctctaa actctttatc accacccaaa acacccgattcctaaaattt 180 cggtacgtaa ggaatcaaat aaactccatg ttcactggca ttgttgaagagattggccga 240 gttaagcaaa tgggttatgg cgaagacggt ggatttcagc ttaaagttgtaggagacatt 300 gtcctaaaag atgtcaatct tggtgacagt atcgcagtta atggtacatgtctaactgtg 360 acggaatttg acactaaagc gtccgaattt actcttggga tagcgcctgagacgcttagg 420 aagacggcat tgatggatct cgaaccaggg tcagttgtta atttagaaagagcccttttg 480 ccttctacac ggatgggtgg tcactttgtc cagggacatg ttgatgggacaggagaaatt 540 gtatcactag ttgaagaagg tgattctttg tgggtcaaga taaaaacaagcccagaaata 600 ctgagataca ttgtaccaaa agggtttatt gcaattgatg gcacaagtttaacagtggtg 660 gatgtgtttg accaagaatt atgctttaat attatgttag ttgcttacactcaacaaaat 720 gtggtcattc cactcaaaaa agttggccaa aaggttaatt tagaggttgatattctagga 780 aaatatgtgg aaaggctcct aagtagtagt ggggttttgg atcctaccaaattcacatag 840 34 279 PRT Spinach 34 Met Ala Leu Ser Thr Ser Leu Ser LeuVal Ser Pro Lys Leu Ser Gln 1 5 10 15 Gln Asn Leu Thr Phe Cys Thr PheAsn Asn Gln Pro Ser Ser Leu Asn 20 25 30 Gly His Ile Lys Phe Asn Pro AsnLeu Arg Asn Ser Val Ser Lys Leu 35 40 45 Phe Ile Thr Thr Gln Asn Thr ArgPhe Leu Lys Phe Arg Tyr Val Arg 50 55 60 Asn Gln Ile Asn Ser Met Phe ThrGly Ile Val Glu Glu Ile Gly Arg 65 70 75 80 Val Lys Gln Met Gly Tyr GlyGlu Asp Gly Gly Phe Gln Leu Lys Val 85 90 95 Val Gly Asp Ile Val Leu LysAsp Val Asn Leu Gly Asp Ser Ile Ala 100 105 110 Val Asn Gly Thr Cys LeuThr Val Thr Glu Phe Asp Thr Lys Ala Ser 115 120 125 Glu Phe Thr Leu GlyIle Ala Pro Glu Thr Leu Arg Lys Thr Ala Leu 130 135 140 Met Asp Leu GluPro Gly Ser Val Val Asn Leu Glu Arg Ala Leu Leu 145 150 155 160 Pro SerThr Arg Met Gly Gly His Phe Val Gln Gly His Val Asp Gly 165 170 175 ThrGly Glu Ile Val Ser Leu Val Glu Glu Gly Asp Ser Leu Trp Val 180 185 190Lys Ile Lys Thr Ser Pro Glu Ile Leu Arg Tyr Ile Val Pro Lys Gly 195 200205 Phe Ile Ala Ile Asp Gly Thr Ser Leu Thr Val Val Asp Val Phe Asp 210215 220 Gln Glu Leu Cys Phe Asn Ile Met Leu Val Ala Tyr Thr Gln Gln Asn225 230 235 240 Val Val Ile Pro Leu Lys Lys Val Gly Gln Lys Val Asn LeuGlu Val 245 250 255 Asp Ile Leu Gly Lys Tyr Val Glu Arg Leu Leu Ser SerSer Gly Val 260 265 270 Leu Asp Pro Thr Lys Phe Thr 275 35 816 DNAarabidopsis 35 atgatggcgg ctcgtactca ttgtatcaac cttatcccca aagtatgtcttccacaatcc 60 ttcagaactg gagaatcagt gactaatctc agatttgatt gcgtctctaagtcatcgaag 120 ctttctctca agacatcatg tggaagatca agaacgcatc accggaggcaaaatctcagc 180 atccggtccg tgtttactgg aatcgtggag gaaatgggtg aagtcaaggacttgggaatg 240 gccgatcacg gaggattcga cctcaaaatc ggagcgagag tggtgttagaggacgtgaag 300 ctcggtgaca gtatcgccgt gaacggtact tgtttaacgg tgacggagtttaacgcagag 360 gagttcacag tagggttagc accggagacg ctgagaaaaa catcgttggaggagttaaag 420 aaaggatctc cggtgaatct ggagcgtgcg ttgcagccag tgagcaggatgggtggacac 480 gtggttcagg gacacgtgga tgggacggga gtgattgaat caatggaggtagagggtgat 540 tctttgtggg tgaaggtgaa agctgacaag ggtttgttga aatacattgtgcctaaagga 600 tttgtggctg ttgatgggac tagcttgacg gttgttgatg tgtttgatgaagagagctgt 660 ttcaatttca tgatgattgc ttatacgcaa cagaatgtag tgattccgactaagaagatt 720 gggcagaaag tgaatcttga ggttgatatc atggggaagt atgttgagaggcttctcacc 780 agtggtggct tctccaaagg aaaagaaaat atttga 816 36 271 PRTarabidopsis 36 Met Met Ala Ala Arg Thr His Cys Ile Asn Leu Ile Pro LysVal Cys 1 5 10 15 Leu Pro Gln Ser Phe Arg Thr Gly Glu Ser Val Thr AsnLeu Arg Phe 20 25 30 Asp Cys Val Ser Lys Ser Ser Lys Leu Ser Leu Lys ThrSer Cys Gly 35 40 45 Arg Ser Arg Thr His His Arg Arg Gln Asn Leu Ser IleArg Ser Val 50 55 60 Phe Thr Gly Ile Val Glu Glu Met Gly Glu Val Lys AspLeu Gly Met 65 70 75 80 Ala Asp His Gly Gly Phe Asp Leu Lys Ile Gly AlaArg Val Val Leu 85 90 95 Glu Asp Val Lys Leu Gly Asp Ser Ile Ala Val AsnGly Thr Cys Leu 100 105 110 Thr Val Thr Glu Phe Asn Ala Glu Glu Phe ThrVal Gly Leu Ala Pro 115 120 125 Glu Thr Leu Arg Lys Thr Ser Leu Glu GluLeu Lys Lys Gly Ser Pro 130 135 140 Val Asn Leu Glu Arg Ala Leu Gln ProVal Ser Arg Met Gly Gly His 145 150 155 160 Val Val Gln Gly His Val AspGly Thr Gly Val Ile Glu Ser Met Glu 165 170 175 Val Glu Gly Asp Ser LeuTrp Val Lys Val Lys Ala Asp Lys Gly Leu 180 185 190 Leu Lys Tyr Ile ValPro Lys Gly Phe Val Ala Val Asp Gly Thr Ser 195 200 205 Leu Thr Val ValAsp Val Phe Asp Glu Glu Ser Cys Phe Asn Phe Met 210 215 220 Met Ile AlaTyr Thr Gln Gln Asn Val Val Ile Pro Thr Lys Lys Ile 225 230 235 240 GlyGln Lys Val Asn Leu Glu Val Asp Ile Met Gly Lys Tyr Val Glu 245 250 255Arg Leu Leu Thr Ser Gly Gly Phe Ser Lys Gly Lys Glu Asn Ile 260 265 27037 603 DNA Magnaporthe grisea 37 atgcacacca aaggcccgac cccgcagcagcacgacggct ccgccctgcg catcggcatc 60 gtgcacgcgc gctggaacga gaccatcatcgagccgcttc tggccggcac aaaagccaag 120 ctgctggcct gcggcgtcaa ggagtccaacatagtcgtgc agagcgttcc ggggtcgtgg 180 gagctgccaa tagccgtgca gaggctctactccgcatccc agctccaaac cccaagctcc 240 ggcccatctc tgtcggccgg cgacctgctcggctcctcga ccacagatct taccgcgctc 300 ccgaccacca ctgcctcatc caccggcccctttgacgccc tcatcgccat cggcgtgcta 360 atcaagggcg agacgatgca ctttgagtacattgccgatt cggtctcgca cggcctgatg 420 cgcgtacagc tcgacacggg cgtcccagttatcttcggcg tcctaacagt cctgaccgac 480 gaccaggcca aggctcgtgc cggcgtcatcgagggcagcc acaaccacgg cgaggactgg 540 ggcctggccg ccgttgagat gggtgtgcgcaggagggatt gggctgccgg gaagaccgag 600 tga 603 38 200 PRT Magnaporthegrisea 38 Met His Thr Lys Gly Pro Thr Pro Gln Gln His Asp Gly Ser AlaLeu 1 5 10 15 Arg Ile Gly Ile Val His Ala Arg Trp Asn Glu Thr Ile IleGlu Pro 20 25 30 Leu Leu Ala Gly Thr Lys Ala Lys Leu Leu Ala Cys Gly ValLys Glu 35 40 45 Ser Asn Ile Val Val Gln Ser Val Pro Gly Ser Trp Glu LeuPro Ile 50 55 60 Ala Val Gln Arg Leu Tyr Ser Ala Ser Gln Leu Gln Thr ProSer Ser 65 70 75 80 Gly Pro Ser Leu Ser Ala Gly Asp Leu Leu Gly Ser SerThr Thr Asp 85 90 95 Leu Thr Ala Leu Pro Thr Thr Thr Ala Ser Ser Thr GlyPro Phe Asp 100 105 110 Ala Leu Ile Ala Ile Gly Val Leu Ile Lys Gly GluThr Met His Phe 115 120 125 Glu Tyr Ile Ala Asp Ser Val Ser His Gly LeuMet Arg Val Gln Leu 130 135 140 Asp Thr Gly Val Pro Val Ile Phe Gly ValLeu Thr Val Leu Thr Asp 145 150 155 160 Asp Gln Ala Lys Ala Arg Ala GlyVal Ile Glu Gly Ser His Asn His 165 170 175 Gly Glu Asp Trp Gly Leu AlaAla Val Glu Met Gly Val Arg Arg Arg 180 185 190 Asp Trp Ala Ala Gly LysThr Glu 195 200 39 9 PRT Artificial Sequence Description of ArtificialSequencePEPTIDE 39 Ala Ser Leu Phe Glu His His Leu Lys 1 5

What is claimed is:
 1. An isolated nucleic acid fragment encoding afungal riboflavin synthase enzyme selected from the group consisting of:(a) an isolated nucleic acid fragment encoding the amino acid sequenceset forth in SEQ ID NO:12; (b) an isolated nucleic acid fragment thathybridizes with (a) under the following hybridization conditions:0.1×SSC, 0.1% SDS, 65° C.; or (c) an isolated nucleic acid fragment thatis complementary to (a) or (b).
 2. There isolated nucleic acid fragmentof claim 1 as set forth in SEQ ID NO:11.
 3. The isolated nucleic acidfragment of claim 1 encoding a fungal riboflavin synthase enzymeobtained from Magnaporthe grisea.